Short-chain nucleic acid elongation primer set, assay kit, and short-chain nucleic acid elongation, amplification and detection methods

ABSTRACT

According to one embodiment, a primer set for elongating a target short-chain nucleic acid containing a first sequence to obtain an elongated product is provided. The elongated product contains a second, a third, a fourth sequence, a complementary sequence of the 1′-th sequence and a sixth sequence. The complementary sequence of the 1′-th sequence is a loop primer sequence. The primer set contains a first elongation primer containing a first elongation primer sequence and a complementary sequence of the sixth sequence, and a second elongation primer containing a second elongation primer sequence, the fourth, the third, and the second sequence.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application a continuation application of U.S. application Ser. No.16/017,367 filed Jun. 28, 2018, now allowed and is based upon and claimsthe benefit of priority from Japanese Patent Applications No.2017-135726, filed Jul. 11, 2017; and No. 2018-083156, filed Apr. 24,2018, the entire contents of all of which are incorporated herein byreference.

FIELD

Embodiments described herein relate generally to a short-chain nucleicacid elongation primer set, an assay kit, and short-chain nucleic acidelongation, amplification and detection methods.

BACKGROUND

In various research fields, attention has been recently focused onshort-chain RNA such as small interference RNA (siRNA) or microRNA(miRNA). siRNA is an artificially synthesized double stranded RNA of 21to 23 bases and is used to suppress gene expression in vivo. It is knownthat miRNA is a single stranded RNA of about 17 to 25 bases, is presentin cells, and has a function to regulate gene expression. Particularly,many reports have been made on the relationship between various diseasesincluding cancer and the kind and expression level of miRNA. Further,since miRNA embedded in the exosome is present in serum, it is expectedas a non-invasive diagnostic tool.

Such short-chain nucleic acid fragments are normally detected byNorthern blotting, microarray analysis or real-time PCR. However, thesequence of the short-chain nucleic acid is short, it is difficult toensure the primer annealing region. Therefore, there is difficulty inamplification, detection or sensitivity improvement. In order toclinically use the short-chain nucleic acid as a diagnostic tool, thedevelopment of the technology of detecting the short-chain nucleic acidis required.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pattern diagram illustrating an example of each of a targetshort-chain nucleic acid, an elongated product, and a short-chainnucleic acid elongation primer set of the first embodiment.

FIG. 2A is a pattern diagram illustrating an example of a step ofobtaining an elongated intermediate product of the first embodiment.

FIG. 2B is a pattern diagram illustrating an example of a step ofobtaining an elongated product of the first embodiment.

FIG. 3 is a pattern diagram illustrating an example of each of anelongated product and a short-chain nucleic acid elongation primer setof the first embodiment.

FIG. 4 is a pattern diagram illustrating an example of each of anelongated product and a short-chain nucleic acid elongation primer setof the first embodiment.

FIG. 5 is a pattern diagram illustrating an example of each of anelongated product and a short-chain nucleic acid elongation primer setof the first embodiment.

FIG. 6 is a flow chart illustrating an example of a short-chain nucleicacid elongation method of the first embodiment.

FIG. 7A is a pattern diagram illustrating an example of each of anelongated product and a short-chain nucleic acid detection primer set ofthe first embodiment.

FIG. 7B is a pattern diagram illustrating an example of each of anelongated product and a short-chain nucleic acid detection primer set ofthe first embodiment.

FIG. 8A is a pattern diagram illustrating an example of each of anelongated product and a short-chain nucleic acid detection primer set ofthe first embodiment.

FIG. 8B is a pattern diagram illustrating an example of each of anelongated product and a short-chain nucleic acid detection primer set ofthe first embodiment.

FIG. 9A is a pattern diagram illustrating an example of each of anelongated product and a short-chain nucleic acid detection primer set ofthe first embodiment.

FIG. 9B is a pattern diagram illustrating an example of each of anelongated product and a short-chain nucleic acid detection primer set ofthe first embodiment.

FIG. 10 is a flow chart illustrating an example of a short-chain nucleicacid amplification method of the first embodiment.

FIG. 11 is a flow chart illustrating an example of a short-chain nucleicacid amplification method of the first embodiment.

FIG. 12A is a pattern diagram illustrating an example of each of anelongated product and a short-chain nucleic acid detection primer set ofthe second embodiment.

FIG. 12B is a pattern diagram illustrating an example of each of anelongated product and a short-chain nucleic acid detection primer set ofthe second embodiment.

FIG. 13A is a pattern diagram illustrating an example of each of anelongated product and a short-chain nucleic acid detection primer set ofthe second embodiment.

FIG. 13B is a pattern diagram illustrating an example of each of anelongated product and a short-chain nucleic acid detection primer set ofthe second embodiment.

FIG. 14A is a pattern diagram illustrating an example of each of anelongated product and a short-chain nucleic acid detection primer set ofthe third embodiment.

FIG. 14B is a pattern diagram illustrating an example of each of anelongated product and a short-chain nucleic acid detection primer set ofthe third embodiment.

FIG. 15A is a pattern diagram illustrating an example of each of anelongated product and a short-chain nucleic acid detection primer set ofthe third embodiment.

FIG. 15B is a pattern diagram illustrating an example of each of anelongated product and a short-chain nucleic acid detection primer set ofthe third embodiment.

FIG. 16 is a pattern diagram of a short-chain nucleic acid detectionprimer set I used in Example 1.

FIG. 17 is a graph showing experimental results in Example 1.

FIG. 18 is a pattern diagram of a short-chain nucleic acid detectionprimer III set used in Example 2.

FIG. 19 is a graph showing experimental results in Example 2.

FIG. 20 is a pattern diagram of a short-chain nucleic acid detectionprimer set V used in Example 3.

FIG. 21 is a graph showing experimental results in Example 3.

FIG. 22 is a pattern diagram of a short-chain nucleic acid detectionprimer set VI used in Example 4.

FIG. 23 is a graph showing experimental results in Example 4.

DETAILED DESCRIPTION

In general, according to one embodiment, a short-chain nucleic acidelongation primer set is for elongating a target short-chain nucleicacid containing a first sequence to obtain an elongated product. Theelongated product is a mutually complementary double-stranded nucleicacid, and one chain contains a second sequence, a third sequence, afourth sequence, a sequence complementary to the complementary sequenceof the first sequence (complementary sequence of the 1′-th sequence),and a sixth sequence in this order in a 3′ to 5′ direction. Thecomplementary sequence of the 1′-th sequence is a loop primer sequence.The short-chain nucleic acid elongation primer set includes a firstelongation primer containing a first elongation primer sequence whichhybridizes with the first sequence and a complementary sequence of thesixth sequence in this order in the 3′ to 5′ direction and a secondelongation primer containing a second elongation primer sequence whichhybridizes with the 1′-th sequence, the fourth sequence, the thirdsequence, and the second sequence in this order in the 3′ to 5′direction.

Hereinafter, various embodiments will be described with reference to thedrawings. Each of the drawings is a pattern diagram for promotingexplanation of the embodiments and their understanding. Although theshape, size, and ratio thereof are different from the actual shape,size, and ratio, the shape, size, and ratio thereof can be appropriatelydesigned and changed taking into consideration the followingdescriptions and known technology.

First Embodiment

Short-Chain Nucleic Acid Elongation Primer Set

The short-chain nucleic acid elongation primer set according to theembodiment is a primer set which elongates a target short-chain nucleicacid in a sample to obtain an elongated product. For example, theelongated product is amplified, whereby the target short-chain nucleicacid in the sample can be detected or quantified.

FIG. 1 shows an example of each of the target short-chain nucleic acid,the elongated product, and the short-chain nucleic acid elongationprimer set of the embodiment. A target short-chain nucleic acid (i) is ashort-chain nucleic acid which is elongated using the short-chainnucleic acid elongation primer set in the short-chain nucleic acidelongation method of the embodiment to be described below. At least apart of the target short-chain nucleic acid (i) may be a nucleic acidcontaining a base sequence. The target short-chain nucleic acid is, forexample, DNA, RNA, PNA, LNA, S-oligo or methyl phosphonate oligo. Thetarget short-chain nucleic acid (i) may be a naturally occurring nucleicacid, or may be a partially or thoroughly artificially synthesizedand/or designed nucleic acid. The target short-chain nucleic acid (i)has, for example, a length of about 50 bases or less.

For example, the target short-chain nucleic acid (i) is, for example, ashort-chain RNA. The short-chain RNA may be, for example, a functionalRNA, such as microRNA (miRNA), a small interfering RNA (siRNA), a smallnuclear RNA (snRNA) or a small nucleolar RNA (snoRNA), or may not be afunctional RNA. Alternatively, the short-chain RNA may be an artificialRNA or an RNA generated by fragmentation of RNA longer than 50 bases.

The target short-chain nucleic acid (i) contains a first sequence. Asillustrated in FIG. 1 , the first sequence is a consecutive sequenceselected from the sequence over the entire length of the targetshort-chain nucleic acid (i). The first sequence may be an entiresequence of the target short-chain nucleic acid (i). The first sequenceis a sequence which can serve as an indicator to detect or quantify thetarget short-chain nucleic acid (i). For example, the first sequence ispreferably a sequence specific to the target short-chain nucleic acid(i) which is contained in the target short-chain nucleic acid (i). Thefirst sequence is a sequence which should be elongated by theshort-chain nucleic acid elongation primer set. It is preferable thatthe first sequence has, for example, a length of 3 to 10 bases, a lengthof from 10 to 20 bases, a length of from 20 to 30 bases, a length offrom 30 to 40 bases or a length of from 40 to 50 bases. It is morepreferable that the first sequence has a length of 10 to 50 bases. Notethat a nucleic acid sequence having the same base sequence as that ofcomplementary DNA (cDNA) of the first sequence (hereinafter, “acomplementary sequence of the first sequence” referred to as “1′-thsequence”) and the complementary sequence thereof should be amplified inthe short-chain nucleic acid amplification method and the detectionmethod of the embodiment to be described below.

Subsequently, the elongated product (ii) obtained by elongating thetarget short-chain nucleic acid (i) using the short-chain nucleic acidelongation primer set of the embodiment is described. The elongatedproduct (ii) is a mutually complementary double-stranded nucleic acid.One chain of the elongated product contains the second sequence, thethird sequence, the fourth sequence, the complementary sequence of the1′-th sequence, the fifth sequence, and the sixth sequence in this orderin the 3′ to 5′ direction. The other chain is a complementary sequenceof the one chain and contains the complementary sequence of the secondsequence, the complementary sequence of the third sequence, thecomplementary sequence of the fourth sequence, the 1′-th sequence, thecomplementary sequence of the fifth sequence, and the complementarysequence of the sixth sequence in the 3′ to 5′ direction.

The 1′-th sequence is a nucleic acid sequence which has the same basesequence as that of the complementary DNA of the first sequence. The1′-th sequence may be, for example, the complementary DNA of the firstsequence. Alternatively, the 1′-th sequence may be a nucleic acidsequence in which at least one nucleotide of the complementary DNA ofthe first sequence is replaced with a locked nucleic acid (LNA) and/or apeptide nucleic acid (PNA) having the same kind of base of thenucleotide.

The complementary sequence of the 1′-th sequence is a nucleic acidsequence having a base sequence corresponding to the base sequence ofthe first sequence. For example, when the target short-chain nucleicacid is an RNA, the base sequence of the complementary sequence of the1′-th sequence is the same as the first sequence except that uracil (U)is thymine (T). Further, for example, when the target short-chainnucleic acid is a DNA, LNA or PNA, the base sequence of thecomplementary sequence of the 1′-th sequence is the same as the firstsequence. The complementary sequence of the 1′-th sequence may beconfigured to be formed by only the DNA, or may contain the LNA and/orPNA.

Each of the second, third, fourth, fifth, and sixth sequences is anucleic acid sequence having a base sequence different from the 1′-thsequence and the complementary sequence thereof. Further, each of thesecond, third, fourth, fifth, and sixth sequences has a mutuallydifferent base sequence. Each of the second, third, fourth, fifth, andsixth sequences may be configured to be formed by only the DNA, or maycontain the LNA and/or PNA. For example, each of these sequences haspreferably a length of 5 to 35 bases and more preferably a length offrom 10 to 30 bases.

The base sequence of each of the second, third, fourth, fifth, and sixthsequences is preferably an artificial sequence. The artificial sequenceis a base sequence which does not exist in nature. These sequences areartificial sequences, thereby preventing the sequences from binding tonucleic acids other than the target short-chain nucleic acid containedin the sample when producing the elongated product using the short-chainnucleic acid elongation primer set to be described below. This resultsin preventing the elongated product containing sequences other than thetarget short-chain nucleic acid from being produced. Thus, it ispossible to prevent the detection result from being false positive.

The artificial sequence can be easily produced by, for example, creatingfour kinds of random number sequences and allocating each of the numbersto adenine (A), guanine (G), cytosine (C) or thymine (T). Further, itmay be confirmed whether the thus produced artificial sequence exists innature by BLAST search or the like. Then, sequences having no sequencethat is the same as or similar to the artificial sequence are selectedas the second sequence, the third sequence, the fourth sequence, thefifth sequence, and the sixth sequence of the embodiment, therebyobtaining a more preferable artificial sequence.

Further, as the second, third, fourth, fifth, and sixth sequences,desired artificial sequences which are efficient as primer recognitionsequences for LAMP may be produced or selected. In that case, thestability of amplification and the specificity during amplification ofthe elongated product are further improved.

Any one of the third sequence, the complementary sequence of the 1′-thsequence, and the fifth sequence is a loop primer sequence or a loopprimer recognition sequence. The loop primer recognition sequence is asequence to which the loop primer contained in the LAMP primer set to bedescribed below is bound when amplifying the elongated product. The loopprimer sequence is a complementary sequence of a loop primer recognitionsequence. The loop primer sequence or the loop primer recognitionsequence is contained, whereby the elongated product is rapidly andstably amplified.

One chain of the elongated product may contain a sequence except thesecond sequence, the third sequence, the fourth sequence, thecomplementary sequence of the first sequence, the fifth sequence, andthe sixth sequence. For example, a spacer sequence may be presentbetween the sequences of each of the second, third, fourth, thecomplementary sequence of the 1′-th, fifth, and sixth sequences. Thespacer sequence will be described for explanation of the firstelongation primer and the second elongation primer.

The elongated product is obtained by elongating the first sequence usingthe first elongation primer and the second elongation primer which arecontained in the short-chain nucleic acid elongation primer set to bedescribed below. Accordingly, the configuration of the elongatedproduct, i.e., the kind of nucleic acid and the base sequence aredetermined according to the configurations of the first elongationprimer and second elongation primer.

The short-chain nucleic acid elongation primer set contains a firstelongation primer (iii) and a second elongation primer (iv).

The first elongation primer (iii) is a single-stranded nucleic acid, andcontains the first elongation primer sequence, the complementarysequence of the fifth sequence, and the complementary sequence of thesixth sequence in this order in the 3′ to 5′ direction.

The first elongation primer sequence is a nucleic acid sequence whichhybridizes with the first sequence and serves as a primer for producingthe complementary sequence (1′-th sequence) over the entire length ofthe first sequence. The first elongation primer sequence has, forexample, at least five consecutive base sequences containing the 5′ endof the 1′-th sequence. This sequence can hybridize with a consecutivebase sequence containing the 3′ end of the first sequence of the targetshort-chain nucleic acid.

The first elongation primer sequence may be configured to be formed byonly the DNA, or may contain the LNA and/or PNA. The amount of LNAand/or PNA contained in the first elongation primer sequence isincreased, thereby strengthening the binding force between the firstelongation primer sequence and the first sequence. Therefore, the numberof LNA and/or PNA to be contained may be determined according to adesired Tm value of the hybridization between the first sequence and thefirst elongation primer sequence. For example, the Tm value is increasedby allowing the first elongation primer sequence to contain the LNAand/or PNA, whereby the first elongation reaction (described below) tobe described below, which hybridizes the first sequence with the firstelongation primer sequence to obtain an elongated intermediate product,can be performed at a higher temperature. Accordingly, it is possible tosuppress a nonspecific binding and the complementary sequence of thefirst sequence is more specifically formed, and thus this is preferred.As a result, it is possible to detect the target short-chain nucleicacid with high accuracy.

For example, the first elongation primer sequence has preferably alength of from 5 to 20 bases, more preferably a length of from 6 to 15bases, and still more preferably a length of from 7 to 12 bases.

The complementary sequence of the fifth sequence is a nucleic acidsequence complementary to the fifth sequence as described above. Thecomplementary sequence of the sixth sequence is a nucleic acid sequencecomplementary to the sixth sequence as described above. Thecomplementary sequence of the fifth sequence and the complementarysequence of the sixth sequence may be configured to be formed by onlythe DNA, or may contain the LNA and/or PNA.

A spacer sequence may be present between the first elongation primersequence and the complementary sequences of the fifth sequence and aspacer sequence may be present between the complementary sequence of thefifth sequence and the complementary sequences of the sixth sequence.The spacer sequence is a nucleic acid sequence which is different fromthe second, third, fourth, 1′-th, fifth, and sixth sequences and thecomplementary sequences thereof and has no adverse effect on theamplification of the elongated product to be described below. The spacersequence may be configured to be formed by only the DNA, or may containthe LNA and/or PNA. For example, the spacer sequence is preferably apoly T or poly A sequence or the like in order to prevent the firstelongation primer from forming a secondary structure. For example, thespacer sequence preferably has a length of from 1 to 16 bases. However,it is preferable that the spacer sequence is not contained in the firstelongation primer, because the first elongation primer can be designedto be shorter.

The first elongation primer preferably has an entire length of from 15to 80 bases. The configuration over the entire length of the firstelongation primer, namely, the base sequence may be selected and/ordesigned so as to obtain a desired elongated product. For example, theconfiguration of the elongated product to be obtained is firstdetermined, and then the base sequence of the first elongation primermay be designed according to the configuration.

Subsequently, the second elongation primer (iv) will be explained. Thesecond elongation primer (iv) is a single-stranded nucleic acid, andcontains the second elongation primer sequence, the fourth sequence, thethird sequence, and the second sequence in this order in the 3′ to 5′direction.

The second elongation primer sequence is a nucleic acid sequence whichserves as a primer for producing the complementary sequence over theentire length of the 1′-th sequence. For example, the second elongationprimer sequence has at least five consecutive base sequences containingthe 5′ end of the complementary sequence of the 1′-th sequence. Thissequence can hybridize with a consecutive base sequence containing the3′ end of the 1′-th sequence of the elongated intermediate product.

The second elongation primer sequence may be configured to be formed byonly the DNA, or may contain the LNA and/or PNA. The amount of LNAand/or PNA contained in the second elongation primer sequence isincreased, thereby strengthening the binding force between the secondelongation primer sequence and the 1′-th sequence. Therefore, the numberof LNA and/or PNA to be contained may be determined according to adesired Tm value of the hybridization between the 1′-th sequence and thesecond elongation primer sequence. For example, the Tm value isincreased by allowing the second elongation primer to contain the LNAand/or PNA, whereby the second elongation reaction to be describedbelow, which hybridizes the 1′-th sequence with the second elongationprimer sequence to obtain an elongated product, can be performed at ahigher temperature. Accordingly, it is possible to suppress anonspecific binding, and thus this is preferred. As a result, it ispossible to detect the target short-chain nucleic acid with highaccuracy.

For example, the second elongation primer sequence has preferably alength of from 5 to 25 bases, more preferably a length of from 8 to 21bases, and still more preferably a length of from 11 to 18 bases.

The second sequence, the third sequence, and the fourth sequence are thesame nucleic acid sequences respectively as the second sequence, thethird sequence, and the fourth sequence described above. Each of thesesequences may be configured to be formed by only the DNA, or may containthe LNA and/or PNA.

A spacer sequence may be present between the second elongation primersequence and the second sequence, between the second sequence and thethird sequence, and/or between the third sequence and the fourthsequences. The spacer sequence is a DNA nucleotide sequence which isdifferent from the second, third, fourth, 1′-th, fifth, and sixthsequences and the complementary sequences thereof and has no adverseeffect on the LAMP of the elongated product to be described below. Thespacer sequence may be configured to be formed by only the DNA, or maycontain the LNA and/or PNA. For example, the spacer sequence ispreferably a poly T or poly A sequence or the like in order to preventthe second elongation primer from forming a secondary structure. Forexample, the spacer sequence preferably has a length of from 1 to 16bases. However, it is preferable that the spacer sequence is notcontained in the second elongation primer, because the second elongationprimer can be designed to be shorter.

In this regard, based on the reason that a binding force (Tm) when thesecond elongation primer hybridizes with the elongated intermediateproduct obtained using the first elongation primer is strengthened, itis preferable that the first extension reaction is performed using theM-MulV enzyme or an enzyme obtained by modifying M-MulV enzyme (e.g.,SuperScript or MultiScribe), thereby allowing a spacer sequence to bepresent between the fourth sequence and the complementary sequence ofthe first sequence. In that case, the spacer sequence is preferably apoly G sequence.

The second elongation primer preferably has an entire length of from 25to 130 bases. The configuration over the entire length of the secondelongation primer, namely, the base sequence may be selected and/ordesigned so as to obtain a desired elongated product. For example, theconfiguration of the elongated product to be obtained is firstdetermined, and then the base sequence of the second elongation primermay be designed according to the configuration.

An example of a step of elongating the target short-chain nucleic acidusing the short-chain nucleic acid elongation primer set as describedabove to obtain an elongated product will be explained with reference toFIGS. 2A and 2B.

As illustrated in FIG. 2A, first, a first elongation primer sequence 4of a first elongation primer 3 hybridizes with a first sequence 2 of atarget short-chain nucleic acid 1. Next, the first DNA polymerase (notshown) to be described below elongates the first elongation primersequence 4 of the 3′ end in the 5′ direction (indicated by an outlinedarrow) using the first sequence 2 as a template. As a result, a 1′-thsequence 5 is produced (hereinafter, a series of the steps is referredto as “first elongation reaction”).

When the target short-chain nucleic acid is an RNA, the first DNApolymerase is a reverse transcriptase in the first elongation reaction,and the 1′-th sequence is produced by reverse transcription of the firstsequence using the first elongation primer.

Then, the target short-chain nucleic acid 1 is dissociated from the1′-th sequence 5, thereby obtaining an elongated intermediate product 6containing the 1′-th sequence 5, the complementary sequence of the fifthsequence, and the complementary sequence of the sixth sequence in thisorder.

After that, as illustrated in FIG. 2B, a second elongation primersequence 9 of a second elongation primer 8 hybridizes with the 1′-thsequence 5 of the elongated intermediate product 6. Thereafter, thesecond DNA polymerase (not shown) to be described below elongates thesecond elongation primer 8 and the elongated intermediate product 6using each other as the template. In other words, the 3′ end of thesecond elongation primer 8 is elongated using the elongated intermediateproduct 6 as a template, and, the 3′ end of the elongated intermediateproduct 6 is elongated using the second elongation primer 8 as atemplate (indicated by an outlined arrow). As a result, an elongatedproduct 11 of double-stranded DNA is produced (hereinafter, a series ofthe steps illustrated in FIG. 2B is referred to as “second elongationreaction”).

As explained above, according to the short-chain nucleic acid elongationprimer set of the first embodiment, an elongated product ofdouble-stranded nucleic acid containing six sequences is obtained usingthe first elongation primer having three sequences and the secondelongation primer having four sequences.

Thus, the sequence length of the first elongation primer is designedshorter than that of the second elongation primer, whereby when thetarget short-chain nucleic acid is an RNA, it is possible to prevent thefirst elongation primer from forming a secondary in the reversetranscription reaction which may be performed at low temperatureaccording to reverse transcriptase characteristics (first elongationreaction). Further, even if the longer second elongation primer has asecondary structure, the secondary structure is disassembled in thesecond elongation reaction to be performed at high temperature, therebyallowing for efficiently performing the second elongation reaction.

Further, the length of each of the base sequences of the first andsecond elongation primers of the first embodiment is designed shorterthan that of the conventional short-chain nucleic acid elongation primerdesigned for LAMP. Thus, secondary structure formation of these primersand the elongated intermediate product hardly occurs compared to theconventional primers. As a result, the efficiency of the above-describedfirst and second elongation reactions is improved, and further anonspecific elongated intermediate product and a nonspecific elongatedproduct are hardly produced. Further, the number of primer recognitionsequences of the elongated product of the embodiment is smaller thanthat of the conventional elongated product for LAMP in which theshort-chain nucleic acid is elongated, but the elongated product of theembodiment contains a loop primer sequence or a loop primer recognitionregion. Thus, it is possible that the elongated product is rapidly andstably amplified.

Further, when the elongated product of the embodiment is amplified,nonspecific amplification hardly occurs, and the elongated product isefficiently amplified even if the abundance of the short-chain nucleicacid is low. Therefore, the short-chain nucleic acid elongation primerset of the embodiment is used so that it is possible to detect andquantify the short-chain nucleic acid with high accuracy.

Each of the sequences included in the above-described short-chainnucleic acid elongation primer set and the elongated product may be arecognition sequence to which the LAMP primer binds. Examples thereofare illustrated in FIGS. 3 to 5 .

Regarding the short-chain nucleic acid elongation primer set and theelongated product to be obtained using the primer set illustrated inFIG. 3 , the second sequence is an F2 sequence, the third sequence is anF1 sequence, the fourth sequence is a B1c sequence, the fifth sequenceis a LB sequence, and the sixth sequence is a B2c sequence.

Therefore, the first elongation primer in this example contains thefirst elongation primer sequence, an LBc sequence, and a B2 sequence inthis order in the 3′ to 5′ direction. The second elongation primercontains the second elongation primer sequence, a B1c sequence, an F1sequence, and an F2 sequence in this order in the 3′ to 5′ direction.Regarding the elongated product, one chain contains the F2, F1, and B1csequences, the complementary sequence of the 1′-th sequence, and the LBand B2c sequences in this order in the 3′ to 5′ direction, and the otherchain contains the F2c, F1c, B1, 1′-th, LBc, and B2 sequences in thisorder in the 3′ to 5′ direction. Here, the F2 sequence is complementaryto the F2c sequence, the F1 sequence is complementary to the F1csequence, the B1 sequence is complementary to the B1c sequence, the B2sequence is complementary to the B2c sequence, and the LB sequence iscomplementary to the LBc sequence (the same holds true for the followingexplanations). In this example, the fifth sequence, i.e., the LBsequence is the loop primer sequence.

According to the short-chain nucleic acid elongation primer set whichhas such a sequence, the short-chain nucleic acid can be elongated morespecifically and efficiently.

Regarding the short-chain nucleic acid elongation primer set and theelongated product to be obtained using the primer set illustrated inFIG. 4 , the second sequence is the F2 sequence, the third sequence isthe F1 sequence, the fourth sequence is the B1c sequence, the fifthsequence is a dummy sequence, and the sixth sequence is the B2csequence.

The dummy sequence is a nucleic acid sequence having a base sequencedifferent from the 1′-th, F2, F1, B1, and B2 sequences and thecomplementary sequences thereof. Further, the dummy sequence is anucleic acid sequence having a base sequence different from any of therecognition sequences contained in the LAMP primer in order to amplifythe elongated product to be described below and the complementarysequences thereof.

Therefore, the first elongation primer in this example contains thefirst elongation primer sequence, a complementary sequence of the dummysequence, and a B2 sequence in this order in the 3′ to 5′ direction. Thesecond elongation primer contains the second elongation primer sequence,a B1c sequence, an F1 sequence, and an F2 sequence in this order in the3′ to 5′ direction. Regarding the elongated product, one chain containsthe F2, F1, and B1c sequences, the complementary sequence of the 1′-thsequence, and the dummy and B2c sequences in this order in the 3′ to 5′direction, and the other chain contains the F2c, F1c, B1, and 1′-thsequences, the complementary sequence of the dummy sequence, and the B2sequence in this order in the 3′ to 5′ direction. In this example, thecomplementary sequence of the 1′-th sequence is the loop primersequence.

According to the short-chain nucleic acid elongation primer set whichhas such a sequence, the loop primer binds to the 1′-th sequence duringamplification of the obtained elongated product, whereby theamplification reaction is accelerated and amplification products arestably produced. Therefore, when the elongation is not correctlyperformed, and the 1′-th sequence and the complementary sequence thereofare not produced, the amplification product is not stably produced. Inother words, when a desired elongated product containing the 1′-thsequence and the complementary sequence thereof is produced, theamplification product is stably obtained and the nonspecific elongatedproduct is hardly amplified. Thus, according to the above-describedshort-chain nucleic acid elongation primer set, the short-chain nucleicacid can be more specifically detected or quantified with high accuracy.

Regarding the short-chain nucleic acid elongation primer set and theelongated product to be obtained using the primer set illustrated inFIG. 5 , the second sequence is an F2 sequence, the third sequence is anLFc sequence, the fourth sequence is an F1 sequence, the fifth sequenceis a B1c sequence, and the sixth sequence is a B2c sequence.

Therefore, the first elongation primer contains the first elongationprimer sequence and B1 and B2 sequences in this order in the 3′ to 5′direction. The second elongation primer contains the second elongationprimer sequence and the F1, LFc, and F2 sequences in this order in the3′ to 5′ direction. Regarding the elongated product, one chain containsthe F2, LFc, and F1 sequences, the complementary sequence of the 1′-thsequence, and the B1c and B2c sequences in this order in the 3′ to 5′direction, and the other chain contains the F2c, LF, F1c, 1′-th, B1, andB2 sequences in this order in the 3′ to 5′ direction. Here, the LFsequence is complementary to the LFc sequence. In this example, thethird sequence, i.e., the LFc sequence is the loop primer recognitionsequence.

According to the short-chain nucleic acid elongation primer set whichhas such a sequence, the short-chain nucleic acid can be elongated morespecifically and efficiently.

The base sequences of the sequences included in the short-chain nucleicacid elongation primer set may be determined based on the base sequencesof the LAMP primer set. For example, according to base sequences in thepre-determined LAMP primer set having excellent amplificationefficiency, the base sequences in the short-chain nucleic acidelongation primer set are determined, whereby the elongated product tobe produced using the short-chain nucleic acid elongation primer set ismore efficiently amplified.

Short Chain Nucleic Acid Elongation Method

Hereinafter, the method of elongating a short-chain nucleic acid usingthe above-described short-chain nucleic acid elongation primer set willbe explained. FIG. 6 is a schematic flow illustrating an example of ashort-chain nucleic acid elongation method of an embodiment. The methodis for elongating a target short-chain nucleic acid containing a firstsequence in a sample, and includes the following steps (S1) to (S3):

hybridizing the first elongation primer with the first sequence andelongating the first sequence to obtain an elongated intermediateproduct containing the 1′-th sequence (S1);

dissociating the elongated intermediate product from the targetshort-chain nucleic acid (S2); and

hybridizing the second elongation primer with the 1′-th sequence of theelongated intermediate product and elongating the second elongationprimer and the elongated intermediate product to obtain an elongatedproduct (S3).

Hereinafter, each step of the short-chain nucleic acid elongation methodwill be explained in detail.

In the step (S1), the first sequence is elongated by hybridizing thefirst elongation primer with the first sequence, and elongating thefirst sequence to obtain an elongated intermediate product containingthe 1′-th sequence. The step (S1) can be performed by, for example,maintaining a first elongation reaction solution containing a sample,the first elongation primer set, and the first DNA polymerase underfirst elongation reaction conditions.

The sample is a sample to be analyzed and may contain the targetshort-chain nucleic acid. Examples of the sample may include substancessuch as blood, serum, white blood cells, lymph fluid, cerebrospinalfluid, urine, feces, sperm, sweat, saliva, mucous membrane in the oralcavity, sputum, tear fluid, mother milk, amniotic fluid, tissue, biopsy,isolated or cultured cells, which are extracted from animals; organs,isolated cells, cultured cells or extracts of plants; mixturescontaining microorganisms, bacteria, fungi or viruses; environmentalsubstances extracted from the environment; mixtures containing syntheticRNA; or materials of any of those mixtures. Alternatively, the samplemay be a preparation obtained by preparing these materials.

The animals may be, for example, mammals, birds, amphibians, reptiles,fishes or arthropods, or may be other organisms belonging to the animalkingdom. The mammals may be any of mammals, for example, primates suchas monkeys and humans, rodents such as mice and rats, companion animalssuch as dogs, cats, and rabbits, livestock such as horses, cows, andpigs.

It is preferable that the sample is in a state which does not block theamplification reaction. The sample is in such a state, whereby thetarget short-chain nucleic acid can be more efficiently amplified in theshort-chain nucleic acid amplification method of the embodiment. Theterm “state which does not block the amplification reaction” means, forexample, a state in which the kind of each of the components in thesample, the combination of the components or the concentration of thecomponents does not reduce, delay or stop the amplification reactionrate.

When each of the above-described material is in the state which does notblock the amplification reaction, the material may be directly used asthe sample. Alternatively, the obtained material is pre-treated by, forexample, any of the known means to obtain a sample in the state whichdoes not block the amplification reaction or in a state more suitablefor amplification. The pretreatment is, for example, chopping,homogenization, centrifugation, precipitation, extraction or separation.

For example, the extraction may be performed using a commerciallyavailable nucleic acid extraction kit. Examples of the nucleic acidextraction kit to be used include ureLink (registered trademark) miRNAIsolation Kit (manufactured by Thermo Fisher Scientific Inc.), microRNAExtractor (registered trademark) SP Kit (manufactured by Wako PureChemical Industries, Ltd.), NucleoSpin (registered trademark) miRNA(manufactured by TAKARA BIO INC.), mirpremier (registered trademark)microRNA Isolarion Kit (manufactured by Sigma-Aldrich Co. LLC.), HighPure miRNA Isolation Kit (manufactured by Roche Life Science), PAXgeneBlood miRNA Kit, miRNeasy Serum/Plasma Kit (both are manufactured byQIAGEN), miRCURY (registered trademark) RNA Isolation Kit-Bifluids(manufactured by Exiqon), and Plasma/Serum RNA Purification Mini Kit(manufactured by Norgen Biotek Corp.), and the nucleic acid extractionkit is not limited thereto. Alternatively, these kits are not used, forexample, a sample may be obtained by diluting materials with a buffersolution, subjecting the resulting diluted solution to heat treatment at80 to 100° C., centrifuging the solution, and extracting thesupernatant.

As the first elongation primer, for example, any of the above-describedfirst elongation primers can be used.

The first DNA polymerase may be any of known DNA polymerases and isselected depending on the kind of the first elongation primer and/or thekind or sequence of the target short-chain nucleic acid. As the firstDNA polymerase, for example, Klenow Fragment (Large Fragment E. coli DNApolymerase I), T4 DNA polymerase, phi29 DNA polymerase, Bst DNApolymerase, Csa DNA polymerase, 96-7 DNA Polymerase, Vent(exo-) DNApolymerase, Deep Vent(exo-) DNA polymerase, GspSSD DNA polymerase or Tinexo-DNA polymerase can be used. Alternatively, other DNA polymeraseswhich are commonly used for PCR amplification (such as Taq DNApolymerase) can be used. Alternatively, reverse transcriptases (such asM-MuLV reverse transcriptase and Transcriptor reverse transcriptase)with which amplification can be performed using DNA as a template can beused.

When the target short-chain nucleic acid is an RNA, the first DNApolymerase is, for example, a reverse transcriptase. As the reversetranscriptase, for example, M-MuLV reverse transcriptase, AMV reversetranscriptase, Transcriptor reverse transcriptase, SuperScript(registered trademark) transcriptor reverse transcriptase, orMultiScribe reverse transcriptase can be used.

When the first elongation primer contains the LNA and/or PNA, aheat-resistant DNA polymerase is preferably used as the first DNApolymerase.

In addition to these components, the first elongation reaction solutionmay further contain a desired component necessary for the firstelongation reaction. The component may be, for example, a salt, asubstrate such as deoxynucleoside triphosphate (dNTPs), a thickener as areaction reagent, a pH adjustment buffer, a surfactant, an ion forincreasing the annealing specificity, and/or an ion serving as acofactor of reverse transcriptase.

The above-described first elongation reaction solution is maintainedunder the first elongation reaction conditions, whereby the firstelongation reaction occurs, and thus it is possible to obtain theabove-described elongated intermediate product containing the 1′-thsequence, the complementary sequence of the fifth sequence, thecomplementary sequence of the sixth sequence. The first elongationreaction conditions may be selected depending on the kind of the firstelongation primer, the kind of the elongated intermediate product and/orthe kind of the DNA polymerase based on common knowledge of thoseskilled in the art. The reaction temperature of the first elongationreaction is, for example, from about 10 to 55° C. The first elongationreaction can be performed by keeping the reaction temperature at aconstant level, keeping a plurality of temperature zones for a certainperiod of time, or repeating the temperature zones in a plurality ofcycles. When the first elongation primer contains the LNA and/or PNA,the reaction temperature of the first elongation reaction is preferablyfrom 20 to 65° C.

For example, the reaction temperature is kept as described above,whereby an elongated intermediate product being bound to the targetshort-chain nucleic acid is obtained in the step illustrated in FIG. 2A.

Then, in the step (S2), the elongated intermediate product isdissociated from the target short-chain nucleic acid. The dissociatingcan be performed by, for example, heating the first elongation reactionsolution to 80 to 100° C. after the first elongation reaction.

When the target short-chain nucleic acid is an RNA, the targetshort-chain nucleic acid is dissociated by the heating, and at the sametime, the reverse transcriptase (first DNA polymerase) may beinactivated. As a result, it is possible to prevent the reversetranscriptase from having an adverse effect on the subsequent stepsafter the step (S3).

Subsequently, in the step (S3), the second elongation primer hybridizeswith the 1′-th sequence of the elongated intermediate product, and thesecond elongation primer and the elongated intermediate product areelongated to obtain an elongated product. The step (S3) can be performedby, for example, maintaining a second elongation reaction solutioncontaining the elongated intermediate product obtained in the step (S2),the second elongation primer, and the second DNA polymerase under secondelongation reaction conditions.

As the second elongation primer, for example, any of the above-describedsecond elongation primer can be used.

The second DNA polymerase may be any of known DNA polymerases and isselected depending on the kind of the second elongation primer and/orthe sequence of the elongated intermediate product. As the second DNApolymerase, Klenow Fragment (Large Fragment E. coli DNA polymerase I),T4 DNA polymerase, phi29 DNA polymerase, Bst DNA polymerase, Csa DNApolymerase, 96-7 DNA Polymerase, Vent(exo-) DNA polymerase, DeepVent(exo-) DNA polymerase, GspSSD DNA polymerase or Tin exo-DNApolymerase can be used. Alternatively, other DNA polymerases which arecommonly used for PCR amplification (such as Taq DNA polymerase) can beused. Alternatively, reverse transcriptases (such as M-MuLV reversetranscriptase and Transcriptor reverse transcriptase) with whichamplification can be performed using DNA as a template can be used. Whenthe first elongation reaction and the second elongation reaction can beperformed by the same kind of DNA polymerase, i.e., when the targetshort-chain nucleic acid is a DNA, LNA, PNA or the like, the first DNApolymerase may be used as the second DNA polymerase. In that case, it isnot necessary to add the second DNA polymerase to the reaction solutionbefore performing the second elongation reaction, and the secondelongation reaction may be performed using a solution obtained by addinga component of the second elongation reaction solution except for DNApolymerase to the first elongation reaction solution.

When the second elongation primer contains the LNA and/or PNA, aheat-resistant DNA polymerase is preferably used as the second DNApolymerase.

In addition to these components, the second elongation reaction solutionmay further contain a desired component necessary for the secondelongation reaction. The component may be, for example, a salt, asubstrate such as dNTPs, a thickener as a reaction reagent, a pHadjustment buffer, a surfactant, an ion for increasing the annealingspecificity, and/or an ion serving as a cofactor of reversetranscriptase.

The above-described second elongation reaction solution is maintainedunder the elongation reaction conditions, whereby the second elongationreaction occurs, and thus it is possible to obtain the elongatedproduct. The elongated product is a mutually complementarydouble-stranded nucleic acid, and one chain contains the second, third,and fourth sequences, the complementary sequence of the 1′-th sequence,and the fifth and sixth sequences in this order in the 3′ to 5′direction. Any one of the third sequence, the complementary sequence ofthe 1′-th sequence, and the fifth sequence is a loop primer sequence ora loop primer recognition sequence.

The second elongation reaction conditions may be selected depending onthe kind of the second elongation primer, the kind of the elongatedintermediate product and/or the kind of the DNA polymerase based oncommon knowledge of those skilled in the art. The reaction temperatureof the second elongation reaction is, for example, from about 10 to 80°C. The second elongation reaction can be performed by keeping thereaction temperature at a constant level, keeping a plurality oftemperature zones for a certain period of time, or repeating thetemperature zones in a plurality of cycles. When the second elongationprimer contains the LNA and/or PNA, the reaction temperature of thesecond elongation reaction is preferably from 20 to 90° C.

For example, the reaction temperature is kept as described above,whereby, for example, an elongated product is obtained in the stepillustrated in FIG. 2B.

The above-described first and second elongation reactions can beperformed in a reaction solution serving both as the first elongationreaction solution and as the second elongation reaction solution. Inother words, for example, before performing the first elongationreaction, a component necessary for the second elongation reaction maybe previously contained in the first elongation reaction solution. Or,after the first elongation reaction, a component necessary for thesecond elongation reaction is added to the reaction solution and usedfor the next reaction. Alternatively, after the first elongationreaction, the whole or part of the reaction solution is added to thesolution containing a component necessary for the second elongationreaction, and the resulting mixture may be used as the second elongationreaction solution. For example, the first elongation primer and thesecond elongation primer may be previously contained in the firstelongation reaction solution. Or the steps (S1) and (S2) are performedusing the first elongation reaction solution not containing the secondelongation primer, and the second elongation primer may be contained inthe reaction solution before the step (S3).

According to the short-chain nucleic acid elongation method, theshort-chain nucleic acid can be elongated more specifically andefficiently.

According to another embodiment, there is provided a method ofelongating a plurality of kinds of the target short-chain nucleic acidshaving different base sequences in a sample. In that case, the first ton-th short-chain nucleic acid elongation primer sets are used. The firstto n-th short-chain nucleic acid elongation primer sets respectivelyinclude the 1₁-th to 1_(n)-th elongation primers for elongating the1₁-th to 1_(n)-th sequences respectively contained in the first to n-thtarget short-chain nucleic acids, and the 2₁-th to 2_(n)-th elongationprimers. Here, n is an integer of 2 or more.

In this case, for example, the one sample is divided into n aliquotes,the 1₁-th to 1_(n)-th elongation primers are respectively added to thealiquotes to prepare the 1₁-th to 1_(n)-th reaction solutions, and afterperforming the first elongation reaction, the second elongation reactionmay be performed using the 2₁-th to 2_(n)-th elongation primers in eachof the reaction solutions. Or a first elongation reaction solutioncontaining the sample and all the 1₁-th to 1_(n)-th elongation primersis prepared, the reaction solution is divided into n aliquotes afterperforming the first elongation reaction, the 2₁-th to 2_(n)-thelongation primers are added to each of the reaction solutions toprepare the 2₁-th to 2_(n)-th reaction solutions, and the secondelongation reaction may be performed. Alternatively, in a firstelongation reaction solution containing the sample and the 1₁-th to1_(n)-th elongation primers, the first elongation reaction is performed,and the second elongation reaction may be performed using the 2₁-th to2_(n)-th elongation primers in the reaction solution without dividingthe reaction solution. Although details will be described below, whenthe reaction solution is not divided in the above manner, the n kinds ofthe target sequences can be distinguished and detected by dividing thereaction solution into n aliquotes in the subsequent step of amplifyingthe elongated product, or using an electrochemical detection device.

According to the short-chain nucleic acid elongation method, a pluralityof kinds of the short-chain nucleic acids can be elongated morespecifically and efficiently.

Short-Chain Nucleic Acid Detection Primer Set

Subsequently, the short-chain nucleic acid detection primer setincluding the short-chain nucleic acid elongation primer set and theLAMP primer set will be explained. The short-chain nucleic aciddetection primer set is a primer set which elongates the targetshort-chain nucleic acid and amplifies the obtained elongated product.

The term “amplification” used herein means that the template nucleicacid is continuously replicated using the primer set and enzymes. Theamplification method to be used in the embodiment is a loop-mediatedisothermal amplification method (LAMP).

The short-chain nucleic acid detection primer set includes theshort-chain nucleic acid elongation primer set and the LAMP primer set.The short-chain nucleic acid elongation primer set is any of theabove-described short-chain nucleic acid elongation primer sets, andincludes the first and second elongation primers. The LAMP primer setincludes a FIP primer, a BIP primer, and a loop primer.

FIGS. 7A to 9B illustrate examples of association of the short-chainnucleic acid detection primer set with the elongated product.

FIG. 7A illustrates an example in which the fifth sequence of theelongated product (ii) is the loop primer sequence. The short-chainnucleic acid elongation primer set is the short-chain nucleic acidelongation primer set of FIG. 1 . The LAMP primer set includes a FIPprimer (v), a BIP primer (vi), and a loop primer (vii). The FIP primer(v) contains complementary sequences of the second and third sequencesin this order in the 3′ to 5′ direction. The BIP primer (vi) containsthe complementary sequence of the sixth sequence and the fourth sequencein this order in the 3′ to 5′ direction. The loop primer (vii) containsthe fifth sequence.

For example, as the short-chain nucleic acid elongation primer in thisexample, the short-chain nucleic acid elongation primer set illustratedin FIG. 3 can be used. FIG. 7B illustrates an example of the short-chainnucleic acid detection primer set in that case. In this example, theLAMP primer set includes the FIP primer containing the F2 and F1csequences in this order in the 3′ to 5′ direction, the BIP primercontaining the B2 and B1c sequences in this order in the 3′ to 5′direction, and the loop primer containing the LB sequence.

According to the short-chain nucleic acid detection primer set, thetarget short-chain nucleic acid can be elongated more specifically andefficiently, and the obtained elongated product can be rapidly andstably amplified.

FIG. 8A illustrates an example in which the complementary sequence ofthe 1′-th sequence of the elongated product (ii) is the loop primersequence. In this example, the FIP primer (v) contains the secondsequences and complementary sequences of the third sequences in thisorder in the 3′ to 5′ direction. The BIP primer (vi) contains thecomplementary sequence of the sixth sequence and the fourth sequence inthis order in the 3′ to 5′ direction. The loop primer (vii) contains acomplementary sequence of the 1′-th sequence.

For example, as the short-chain nucleic acid elongation primer in thisexample, the short-chain nucleic acid elongation primer set illustratedin FIG. 4 can be used. FIG. 8B illustrates an example of the short-chainnucleic acid detection primer set in that case. In this example, theLAMP primer set includes the FIP primer containing the F2 and F1csequences in this order in the 3′ to 5′ direction, the BIP primercontaining the B2 and B1c sequences in this order in the 3′ to 5′direction, and the loop primer containing the complementary sequence ofthe 1′-th sequence.

According to the short-chain nucleic acid detection primer set, thetarget short-chain nucleic acid can be elongated more specifically andefficiently, and the obtained elongated product can be rapidly andstably amplified. Further, the use of the short-chain nucleic aciddetection primer set results in the following condition: when a desiredelongated product containing the 1′-th sequence and the complementarysequence thereof is produced, the loop primer hybridizes with the 1′-thsequence and the amplification product is stably obtained and thenonspecific elongated product is hardly amplified. Therefore, it ispossible to more specifically amplify the short-chain nucleic acid.

FIG. 9A illustrates an example in which the third sequence of theelongated product (ii) is the loop primer recognition sequence. In thisexample, the FIP primer (v) contains complementary sequences of thesecond and fourth sequences in this order in the 3′ to 5′ direction. TheBIP primer (vi) contains the complementary sequence of the sixthsequence and the fifth sequence in this order in the 3′ to 5′ direction.The loop primer (vii) contains a complementary sequence of the thirdsequence.

For example, as the short-chain nucleic acid elongation primer in thisexample, the short-chain nucleic acid elongation primer set illustratedin FIG. 5 can be used. FIG. 9B illustrates an example of the short-chainnucleic acid detection primer set in that case. In this example, theLAMP primer set includes the FIP primer containing the F2 and F1csequences in this order in the 3′ to 5′ direction, the BIP primercontaining the B2 and B1c sequences in this order in the 3′ to 5′direction, and the loop primer containing the LF sequence.

According to the short-chain nucleic acid detection primer set, thetarget short-chain nucleic acid can be elongated more specifically andefficiently, and the obtained elongated product can be rapidly andstably amplified.

Short Chain Nucleic Acid Amplification Method

Hereinafter, the method of amplifying a target short-chain nucleic acidusing the above-described short-chain nucleic acid detection primer setwill be explained.

FIG. 10 is a schematic flow illustrating an example of a short-chainnucleic acid amplification method of an embodiment. The method is amethod of amplifying a target short-chain nucleic acid containing afirst sequence in a sample and includes the following steps (S11) to(S14):

hybridizing the first elongation primer with the first sequence andelongating the first sequence to obtain an elongated intermediateproduct containing the 1′-th sequence (S11);

dissociating the elongated intermediate product from the targetshort-chain nucleic acid (S12);

hybridizing the second elongation primer with the 1′-th sequence of theelongated intermediate product and elongating the second elongationprimer and the elongated intermediate product to obtain an elongatedproduct (S13);

and maintaining an amplification reaction solution containing theelongated product, the LAMP primer set, and a strand displacement DNApolymerase under isothermal amplification reaction conditions, therebyamplifying the 1′-th sequence and/or the complementary sequence thereofusing the elongated product as a template to obtain an amplificationproduct (S14).

First, the steps (S11) to (S13) are executed. The steps (S11) to (S13)can be respectively performed by, for example, the same methods as thoseof the steps (S1) to (S3) of the above-described short-chain nucleicacid elongation method.

In the step (S14), an amplification reaction solution containing theelongated product obtained in the step (S13), the LAMP primer set, and astrand displacement DNA polymerase is maintained under isothermalamplification reaction conditions.

The LAMP primer set is any of the above-described primer sets for LAMP.

The strand displacement DNA polymerase is an enzyme for catalyzing anisothermal amplification reaction which amplifies the 1′-th sequence andthe complementary sequence thereof using the elongated product be atemplate. Although the strand displacement DNA polymerase is notparticularly limited, and examples thereof may include Bst, Bst2.0,Bst3.0, GspSSD, GspM, Tin, Bsm, Csa, 96-7, phi29, OminiAmp (registeredtrademark), Aac, BcaBEST (registered trademark), DisplaceAce (registeredtrademark), SD, StrandDisplace (registered trademark), TOPOTAQ,Isotherm2G, Taq, and a combination thereof. The amplification reactionsolution may further contain a desired component necessary for theamplification reaction, in addition to those components. The componentmay be, for example, a substrate such as deoxynucleoside triphosphate(dNTPs) or a salt compound for maintaining an appropriate amplificationenvironment.

The isothermal amplification reaction conditions may be selecteddepending on the LAMP primer set and the kind of the strand displacementDNA polymerase based on common knowledge of those skilled in the art.

The isothermal amplification reaction conditions include the followings:temperature: 50 to 75° C.; and time: 30 to 90 minutes, wherein thetemperature is preferably from 60 to 70° C.

The amplification reaction solution is maintained under isothermalamplification reaction conditions, thereby amplifying the 1′-th sequenceand/or the complementary sequence thereof using the elongated product asa template to obtain an amplification product containing the 1′-thsequence and/or the complementary sequence thereof.

According to the above-described short-chain nucleic acid amplificationmethod, the target short-chain nucleic acid in the sample can beelongated more specifically and efficiently, and the obtained elongatedproduct can be rapidly and stably amplified. Further, the short-chainnucleic acid detection primer sets illustrated in FIGS. 8A and 8B areused, whereby the target short-chain nucleic acid can be furtherspecifically amplified.

In another embodiment, the second DNA polymerase used in the steps (S3)and (S13) may be a strand displacement DNA polymerase. In that case, thesecond DNA polymerase used in the steps (S3) and (S13) may be used asthe strand displacement DNA polymerase in the step (S14). In that case,for example, after the completion of the second elongation reaction, thestep (S14) may be performed without adding the strand displacement DNApolymerase to the amplification reaction solution. In that case, it ispossible to reduce the amount of the enzyme to be used and cut costs.Further, it is possible to make the experimental procedure simpler.

The above-described first elongation reaction, second elongationreaction, and amplification reaction can be performed in a reactionsolution which serves as the first elongation reaction solution, thesecond elongation reaction solution, and the amplification reactionsolution. In other words, for example, before performing the firstelongation reaction, components necessary for the second elongationreaction and the amplification reaction may be previously contained inthe first elongation reaction solution. Alternatively, after each of thereactions, a component necessary for the next reaction is added to thereaction solution, and the resulting mixture may be used for the nextreaction. Alternatively, after the each reaction, the whole or part ofthe reaction solution may be added to the solution containing acomponent necessary for the next reaction. In that case, the operationis simpler because it is not necessary to perform the following stepsof: removing the nonspecific products produced by the first elongationreaction and the second elongation reaction; separating the elongatedintermediate product from the second elongation reaction; and separatingthe elongated product for the amplification reaction. This is achievedby using the short-chain nucleic acid elongation primer set of theembodiment which can elongate the target short-chain nucleic acidspecifically.

Short-Chain Nucleic Acid Detection Method

In another embodiment, there is provided a target short-chain nucleicacid detection method.

FIG. 11 is a schematic flow illustrating an example of a short-chainnucleic acid detection method of an embodiment. The method is a methodof detecting a target short-chain nucleic acid containing a firstsequence in a sample and includes the following steps (S21) to (S25):

hybridizing the first elongation primer with the first sequence andelongating the first sequence to obtain an elongated intermediateproduct containing the 1′-th sequence (S21);

dissociating the elongated intermediate product from the targetshort-chain nucleic acid (S22);

hybridizing the second elongation primer with the 1′-th sequence of theelongated intermediate product and elongating the second elongationprimer and the elongated intermediate product to obtain an elongatedproduct (S23);

maintaining an amplification reaction solution containing the elongatedproduct, the LAMP primer set, and a strand displacement DNA polymeraseunder isothermal amplification reaction conditions, thereby amplifyingthe 1′-th sequence and/or the complementary sequence thereof using theelongated product as a template to obtain an amplification product(S24); and

detecting the obtained amplification product during maintaining underthe amplification reaction conditions of the step (S24) (S25).

The steps (S21) to (S24) can be respectively performed by, for example,the same methods as those of the steps (S11) to (S14) as describedabove.

In the step (S25), the obtained amplification product is detected duringmaintaining under amplification reaction conditions. The amplificationproduct can be detected using turbidity, an optical signal or anelectrochemical signal as an indicator.

When the amplification product is detected using turbidity as theindicator, the turbidity of the reaction solution may be detected. Theturbid of the reaction solution is caused by, for example, magnesiumpyrophosphate which is produced depending on the amplification productor the presence of the amplification reaction. Therefore, for example,the higher the abundance of the amplification product, the higher theturbidity. The detecting can be performed, for example, using aturbidimeter or an absorption spectrometer or visual observation.

When the amplification product is detected using an optical signal as anindicator, a marker substance which produces an optical signal (e.g., afluorescence reagent containing calcein or an intercalator) can be used.Wherein the optical signal changes depending on the presence of theamplification product or the amplification reaction. The markersubstance is previously contained in the amplification reaction solutionand the optical signal from the marker substance may be detected duringmaintaining under the amplification reaction conditions. The detectingcan be performed, for example, using any of known photo sensors orvisual observation.

When the amplification product is detected using an electrochemicalsignal as an indicator, for example, a marker substance which generatesan electrochemical signal from an oxidation-reduction reaction or thelike can be used. Wherein the electrochemical signal changes dependingon an increase in the amplification product. The marker substance ispreviously contained in the amplification reaction solution, and theelectrochemical signal from the marker substance may be detected duringmaintaining under the amplification reaction conditions. The detectingcan be performed, for example, using the electrochemical detectiondevice including an electrode for detecting the electrochemical signalto be described below.

The electrochemical signal generating marker substance is, for example,an oxidizing agent whose oxidation reduction potential can serve as anelectrochemical signal. Examples of the electrochemical signalgenerating marker substance include ferricyanide ions, ferrocyanideions, iron complex ions, ruthenium complex ions, and cobalt complexions. Each of these marker substances is obtained by dissolvingpotassium ferricyanide, potassium ferrocyanide, an iron complex, aruthenium complex or a cobalt complex in a reaction solution.

For example, when ferricyanide ion (Fe(CN)₆ ⁴⁻) is used as the markersubstance, electrons are released by an oxidation reaction in whichFe(CN)₆ ⁴⁻ is converted to Fe(CN)₆ ³⁻. Each of these marker substancesis rebound to an amplification product having a negative charge and isaway from the amplification product. Thus, in the electrode in which theamplification products are present in the vicinity, a current(electrochemical signal) to be detected with an increase in theamplification product decreases.

Alternatively, the electrochemical signal generating marker substancemay be a redox probe. The redox probe is, for example, a substancehaving an oxidation reduction potential of from −0.5 V to 0.5 V, andelectrostatically binds to an amplification product in a reactionsolution. The redox probe bound to the amplification product is oxidizedor reduced by applying a voltage to the electrode, and the reactionresults in release of electrons. Accordingly, for example, in theelectrode in which the amplification products are present in thevicinity, the current (electrochemical signal) to be detected with anincrease in the amplification product increases or a peak potential ofoxidation-reduction to be detected shifts to a negative direction.Therefore, in addition to the electrochemical signal, the peak potentialof oxidation-reduction is detected, whereby the measurement can beperformed with high accuracy.

The redox probe is, for example, a metal complex. Regarding the metalcomplex used as the redox probe, a central metal is, for example,ruthenium (Ru), rhodium (Rh), platinum (Pt), cobalt (Co), chromium (Cr),cadmium (Cd), nickel (Ni), zinc (Zn), copper (Cu), osmium (Os), iron(Fe) or silver (Ag). The metal complex is, for example, an amminecomplex, a cyano complex, a halogen complex, a hydroxy complex, acyclopentadienyl complex, a phenanthroline complex or a bipyridinecomplex. Further, redox probes such as methylene blue, Nile blue, andcrystal violet can be used.

For example, the electrochemical signal generating marker substance isruthenium hexane amine (RuHex). In that case, when the amplificationproduct is present, RuHex³⁺ bound to the amplification product isreduced to RuHex²⁺ by applying a voltage to the electrode and electronsare released. The electrons flow into the electrode, whereby theamplification product can be detected.

The electrochemical detection device includes a chip. The chip includesa substrate having at least one electrode on one surface. The electrodecan be obtained by, for example, forming a metallic pattern having adesired shape, such as a circular shape or a square shape on asubstrate. It is preferable that the metal is, for example, gold becausethe sensitivity thereof is favorable. When the amplification reactionsolution is brought onto the one surface, the electrode comes intocontact with the amplification reaction solution, thereby detecting theelectrochemical signal from the marker substance in the amplificationreaction solution. The substrate may further include a pad. The pad iselectrically connected to the electrode, and information on theelectrochemical signal obtained from the electrode can be retrieved fromthe pad. Further, the substrate may include a reference electrode and acounter electrode.

The electrochemical detection device may include a measurement unit thatreceives a detection signal from the electrode of the chip, a controlunit that controls the measuring unit, generates measurement data fromthe detected signal, and stores the measurement data in a memory, aquantification unit that quantifies the target short-chain nucleic acidin the sample based on the measurement data, a liquid-feed unit thatfeeds the amplification reaction solution to the chip and takes out itby control by the control unit, and/or a temperature control unit thatcontrols the temperature of the chip by control by the control unit.These units may be computers.

The turbidity, optical signal, and electrochemical signal explainedabove may be detected within a specified time from the start of theamplification reaction or may be temporarily detected. The temporarilymay mean continuously, or may mean to detect intermittently (i.e., at adesired time interval) at a plurality of points of time. In suchdetecting, as the abundance of the target short-chain nucleic acidpresent in the sample is higher, a rise of change of the signal isobserved in a shorter time. For example, the target short-chain nucleicacid can be detected or quantified from the time to threshold in thefollowing manner. A plurality of standard samples containing theshort-chain nucleic acid at different known concentrations is used tocreate a calibration curve of the time to threshold of the detectionsignal relative to the abundance of the short-chain nucleic acid. Then,the calibration curve is compared to the measurement results of the timeto threshold in the target short-chain nucleic acid. Based on this, theabundance of the target short-chain nucleic acid in the sample can becalculated.

In another embodiment of the short-chain nucleic acid detection method,the 1′-th sequence and the complementary sequence thereof are separatedfrom other sequences by fragmenting the amplification product with aspecific restriction enzyme, and the 1′-th sequence and thecomplementary sequence thereof may be detected. In that case, the firstelongation primer, the second elongation primer, and a primer set whichbinds to the elongated product is designed so that the amplificationproduct contains the sequence which can be cut with the specificrestriction enzyme. In that case, after the amplification reaction, theamplification product is treated with the corresponding restrictionenzyme. Then, for example, the amplification product is analyzed byelectrophoresis. When the target short-chain nucleic acid is present inthe sample, and the sequence containing the 1′-th sequence and thecomplementary sequence thereof, as a result of electrophoresis,amplification products appear as a band in a specific position. Based onthis, the presence or absence of the target short-chain nucleic acid inthe sample can be clearly determined.

According to the short-chain nucleic acid detection method of theabove-described embodiment, the target short-chain nucleic acid in thesample is elongated more specifically and efficiently, and the obtainedelongated product can be rapidly and stably amplified. Thus, the targetshort-chain nucleic acid can be detected accurately and specifically.

Further, when the target short-chain nucleic acid is, for example, ashort-chain nucleic acid which is expressed or whose expression levelincreases or decreases in cells with a specific disease, it is possibleto accurately and specifically determine whether the living body fromwhich the sample is collected has the specific disease by detecting thetarget short-chain nucleic acid by the detection method of theembodiment. Examples of the specific disease include cancers such asbreast cancer, colon cancer or lung cancer, and other diseases. Forexample, when the target short-chain nucleic acid is a short-chainnucleic acid which is expressed or whose expression level increases ordecreases in a specific bacterium or virus, it is possible to accuratelyand specifically determine the presence of the specific bacteria orvirus in the sample or to determine whether the living body from whichthe sample is collected is infected with a specific bacterium or virusby detecting the target short-chain nucleic acid by the detection methodof the embodiment.

According to another embodiment, there is provided a method of detectinga plurality of kinds of the target short-chain nucleic acids havingdifferent base sequences contained in one kind of sample. In that case,the first to n-th primer sets for short-chain nucleic acid detection areused. The first to n-th short-chain nucleic acid detection primer setsrespectively include the first to n-th short-chain nucleic acidelongation primer sets for elongating the 1₁-th to 1_(n)-sequencesrespectively contained in the first to n-th target short-chain nucleicacids; and the first to n-th LAMP primer sets for amplifying the firstto n-th elongated products obtained using the first to n-th short-chainnucleic acid elongation primer sets. Here, n is an integer of 2 or more.

For example, when the n kinds of the reaction solutions containing the nkinds of the elongated products respectively are produced in theabove-described short chain nucleic acid elongation method, in each ofthe reaction solutions, the first to n-th amplification products areobtained using the first to n-th LAMP primer sets and the abundance ofeach of amplification products is detected, whereby the n kinds of thetarget sequences can be distinguished and detected.

Alternatively, when one reaction solution containing the n kinds of theelongated products is produced in the above-described short chainnucleic acid elongation method, the n kind of the elongated products canbe distinguished and detected, for example, using the electrochemicaldetection device. The electrochemical detection device includes a chipincluding a substrate with at least n electrodes on one surface. Thefirst to n—the LAMP primer sets are releasably immobilized on the onesurface near each of the electrodes. To the reaction solution containingthe n kinds of the elongated products, any of the above-describedelectrochemical signal producing marker substances is added. Thisreaction solution is brought onto the one surface of a substrate, and areaction solution is maintained on isothermal amplification conditions,whereby, in each of the electrodes, the corresponding elongated productis amplified using the first to n-th LAMP primer sets. Therefore, thetime to threshold of the electrochemical signal of each of theelectrodes is measured, whereby the n kinds of the target short-chainnucleic acids can be detected and quantified.

According to the above method, a plurality of kinds of the targetshort-chain nucleic acids can be detected and quantified accurately andspecifically.

Assay Kit

According to another embodiment, there is provided an assay kit fordetecting the short-chain nucleic acid. The kit includes a short-chainnucleic acid detection primer set, a nucleic acid elongation reagent,and an LAMP reagent.

The short-chain nucleic acid detection primer set contained in the assaykit is any of the above-described short-chain nucleic acid detectionprimer sets. The short-chain nucleic acid elongation primer set includedin the short-chain nucleic acid detection primer set and the LAMP primerset may be accommodated in the separate containers.

The nucleic acid elongation reagent contains the first DNA polymeraseand the second DNA polymerase. The nucleic acid elongation reagent mayfurther contain other components necessary for the above-described firstand second elongation reactions, such as a salt, a substrate such asdeoxynucleoside triphosphate (dNTPs), a thickener as a reaction reagent,a pH adjustment buffer, a surfactant, an ion for increasing theannealing specificity, and/or an ion serving as a cofactor of reversetranscriptase, in addition to those components.

The LAMP reagent contains any of the above-described strand displacementDNA polymerases. The LAMP reagent may contain other components necessaryfor the above-described isothermal amplification reaction, such as asubstrate (e.g., deoxynucleoside triphosphate (dNTPs)) or a saltcompound for maintaining an appropriate amplification environment.

The assay kit may further include a marker substance generating anelectrical signal which changes with an increase in the amplificationproduct, and an electrochemical detection device for detecting theelectrical signal. As the marker substance and the electrochemicaldetection device, for example, the above-described marker substance andelectrochemical detection device can be used.

According to another embodiment, the assay kit may be for detecting aplurality of kinds of the target short-chain nucleic acids havingdifferent base sequences. For example, when the assay kit is fordetecting n-kinds of the target short-chain nucleic acids havingdifferent base sequences, the assay kit includes the first to n-thshort-chain nucleic acid detection primer sets. The first to n-thshort-chain nucleic acid detection primer sets include the first to n-thshort-chain nucleic acid elongation primer sets respectively forelongating the 1₁-th to 1_(n)-sequences contained in the first to n-thtarget short-chain nucleic acids respectively; and the first to n-thLAMP primer sets. Here, n is a natural number.

In the above example, the first to n-th primer sets for short-chainnucleic acid elongation and the first to n-th primer sets for LAMP maybe accommodated in separate containers.

The above assay kit may include a chip including a substrate with atleast n electrodes on one surface and the above-describedelectrochemical detection device in which the first to n-th LAMP primersets are freely immobilized on the one surface near each of theelectrodes.

According to the assay kit, the target short-chain nucleic acid can bedetected and quantified accurately and specifically.

Second Embodiment

In another embodiment, the first elongation primer may not necessarilycontain the fifth sequence. FIGS. 12A to 13B each show an example ofsuch a short chain nucleic acid elongation primer set, an elongationproduct produced with use thereof, and a LAMP primer set for amplifyingan elongation product.

In the example of FIG. 12A, as to an elongation product (ii), one chaincontains the second, third, and fourth sequences, the complementarysequence of the 1′-th sequence, and the sixth sequence in this order inthe 5′ to 3′ direction. The other chain is a complementary sequence ofthe above-mentioned one chain and contains the complementary sequence ofthe second sequence, the complementary sequence of the third sequence,the complementary sequence of the fourth sequence, the 1′-th sequenceand the complementary sequence of the sixth sequence in this order inthe 3′ to 5′ direction. In this example, the complementary sequence ofthe 1′-th sequence is a loop primer sequence.

The short chain nucleic acid elongation primer set contains the firstelongation primer (iii) and the second elongation primer (iv). The firstelongation primer (iii) contains the first elongation primer sequenceand the complementary sequence of the sixth sequence in this order inthe 3′ to 5′ direction. The second elongation primer (iv) contains thesecond elongation primer sequence, the fourth sequence, the thirdsequence and the second sequence in this order in the 3′ to 5′direction.

The LAMP primer set contains an FIP primer (v), a BIP primer (vi) and aloop primer (vii). The FIP primer (v) contains the second sequence andthe complementary sequence of the third sequence in this order in the 3′to 5′ direction. The BIP primer (vi) contains the complementary sequenceof the sixth sequence and the fourth sequence in this order in the 3′ to5′ direction. The loop primer (vii) contains the complementary sequenceof the 1′-th sequence.

Here, as the second sequence, the third sequence, the fourth sequence,the complementary sequence of the 1′-th sequence and the sixth sequence,mentioned above, materials similar to those described in the firstembodiment can be used. Moreover, as in the first embodiment, eachprimer and elongation product may also contain a sequence other thanthose set forth above, for example, a spacer sequence.

FIG. 12B shows an example in which each sequence in the example of FIG.12A is a recognition sequence to which a LAMP primer may hybridize. Inthis example, the second sequence is an F2 sequence, the third sequenceis an F1 sequence, the fourth sequence is a B1c sequence and the sixthsequence is a B2c sequence.

Therefore, as to the elongation product (ii), one chain contains the F2sequence, the F1 sequence, the B1c sequence, the complementary sequenceof the 1′-th sequence and the B2c sequence in this order in the 5′ to 3′direction, whereas the other chain contains the F2c sequence, the F1csequence, the B1 sequence, the 1′-th sequence and the B2 sequence inthis order in the 3′ to 5′ direction. The first elongation primer (iii)contains the first elongation primer sequence and the B2 sequence inthis order in the 3′ to 5′ direction. The second elongation primer (iv)contains the second elongation primer sequence, the B1c sequence, the F1sequence and the F2 sequence in this order in the 3′ to 5′ direction.

The FIP primer (v) contains the F2 sequence and the F1c sequence in thisorder in the 3′ to 5′ direction. The BIP primer (vi) contains the B2sequence and the B1c sequence in this order in the 3′ to 5′ direction.The loop primer (vii) contains the complementary sequence of the 1′-thsequence.

FIG. 13A shows an example in which the sequences of the elongationproduct (ii), the first elongation primer (iii) and the secondelongation primer (iv) are arranged in the same order as that of FIG.12A, but the third sequence is a loop primer recognition sequence.

The FIP primer (v) contains the second sequence and the complementarysequence of the fourth sequence in this order in the 3′ to 5′ direction.The BIP primer (vi) contains the complementary sequence of the sixthsequence and the complementary sequence of the 1′-th sequence in thisorder in the 3′ to 5′ direction. The loop primer (vii) contains thecomplementary sequence of the third sequence.

FIG. 13B shows an example in which each sequence in the example of FIG.13A set forth above is a recognition sequence to which a LAMP primer mayhybridize. In this example, the second sequence is an F2 sequence, thethird sequence is an LFc sequence, the fourth sequence is an F1 sequenceand the sixth sequence is a B2c sequence.

Therefore, as to the elongation product (ii), one chain contains the F2,LFc, F1 sequences, the complementary sequence of the 1′-th sequence andthe B2c sequence in this order in the 5′ to 3′ direction, whereas theother chain contains the F2c, LF, F1c, the 1′-th and B2 sequences inthis order in the 3′ to 5′ direction. The first elongation primer (iii)contains the first elongation primer sequence and the B2 sequence inthis order in the 3′ to 5′ direction. The second elongation primer (iv)contains the second elongation primer sequence, the F1, LFc and F2sequences in this order in the 3′ to 5′ direction.

The FIP primer (v) contains the F2 sequence and the F1c sequence in thisorder in the 3′ to 5′ direction. The BIP primer (vi) contains the B2sequence and the complementary sequence of the 1′-th sequence in thisorder in the 3′ to 5′ direction. The loop primer (vii) contains the LFsequence. In this example, the complementary sequence of the 1′-thsequence functions as a B1c sequence.

As described above, according to the second embodiment, a short chainnucleic acid elongation primer set which does not contain the fifthsequence and also a short chain nucleic acid detection primer set whichcontains the above-described LAMP primer set are provided. Further, anassay kit which contains a short chain nucleic acid detection primer setdescribed in the first embodiment is also provided. Moreover, the shortchain nucleic acid elongation primer set and the short chain nucleicacid detection primer set according to the second embodiment can be usedsimilarly in the short chain nucleic acid elongation method, the shortchain nucleic acid amplification method and the short chain nucleic aciddetection method described in the first embodiment.

According to the second embodiment, the first elongation primer containstwo sequences. Thus, the first elongation primer is shorter, andtherefore when used for the short chain nucleic acid elongation method,it is possible to further prevent the first elongation primer andelongated intermediate product from being formed into a secondarystructure. Moreover, the manufacturing cost of the first elongationprimer can be further reduced.

In addition, the elongated product produced using the short chainnucleic acid elongation primer set of the second embodiment containsless primer recognition sequences in number than the conventionalelongated product for the LAMP, but contains a loop primer sequence or aloop primer recognition region as in the case of the first embodiment.Therefore, with use of the short chain nucleic acid amplification methodand the short chain nucleic acid detection method, it is possible tostably and quickly amplify the elongated product. In addition, even ifthe quantity of short chain nucleic acid is small, it can be amplifiedefficiently. Thus, with use of the short chain nucleic acid detectionprimer set of the second embodiment, it is possible to detect orquantify short chain nucleic acid more precisely.

The first and second elongation primers according to the secondembodiment, if they have such a structure that the complementarysequence of the 1′-th sequence as shown in FIG. 12A or 12B is a loopprimer recognition sequence, a loop primer may hybridize with the 1′-thsequence to produce an amplification product when an elongated productis produced. Therefore, the nonspecific elongated product is hardlyamplified. Thus, it is possible to amplify short chain nucleic acid morespecifically. Moreover, this structure is advantageous because thedesigning of both elongation primers is easier.

The first and second elongation primers according to the secondembodiment, if they have such a structure that the complementarysequence of the 1′-th sequence as shown in FIG. 13A or 13B is a B1csequence, a BIP primer may hybridize with the 1′-th sequence to producean amplification product when an elongated product is produced.Therefore, the nonspecific elongated product is hardly amplified. Thus,it is possible to amplify short chain nucleic acid more specifically.

Third Embodiment

In a further embodiment, the second elongating primer may notnecessarily contain the fourth sequence. FIGS. 14A to 15B each show anexample of such a short chain nucleic acid elongation primer set, anelongation product produced with use thereof, and a LAMP primer set foramplifying an elongated product.

In the example of FIG. 14A, as to the elongated product (ii), one chaincontains the second sequence, the third sequence, the complementarysequence of the 1′-th sequence, the fifth sequence and the sixthsequence in this order in the 5′ to 3′ direction. The other chain is thecomplementary sequence of the above-mentioned one chain and contains thecomplementary sequence of the second sequence, the complementarysequence of the third sequence, the 1′-th sequence, the complementarysequence of the fifth sequence and the complementary sequence of thesixth sequence in this order in the 3′ to 5′ direction. In this example,the fifth sequence is a loop primer sequence.

The first elongation primer (iii) contains the first elongation primersequence, the complementary sequence of the fifth sequence and thecomplementary sequence of the sixth sequence in this order in the 3′ to5′ direction. The second elongation primer (iv) contains the secondelongation primer sequence, the third sequence and the second sequencein this order in the 3′ to 5′ direction.

The LAMP primer set contains an FIP primer (v), a BIP primer (vi) and aloop primer (vii). The FIP primer (v) contains the second sequence, thecomplementary sequence of the third sequence in this order in the 3′ to5′ direction. The BIP primer (vi) contains the complementary sequence ofthe sixth sequence and the complementary sequence of the 1′-th sequencein this order in the 3′ to 5′ direction. The loop primer (vii) containsthe fifth sequence.

Here, as the second sequence, the third sequence, the complementarysequence of the 1′-th sequence, the fifth sequence and the sixthsequence, mentioned above, materials similar to those described in thefirst embodiment can be used. Moreover, as in the first embodiment, eachprimer and elongation product may also contain a sequence other thanthose set forth above, for example, a spacer sequence.

FIG. 14B shows an example in which each sequence in the example of FIG.14A set forth above is a recognition sequence to which a LAMP primer mayhybridize. In this example, the second sequence is an F2 sequence, thethird sequence is an F1 sequence, the fifth sequence is an LB sequenceand the sixth sequence is a B2c sequence.

Therefore, as to the elongated product (ii), one chain contains the F2sequence, the F1 sequence, the complementary sequence of the 1′-thsequence, the LB sequence and the B2c sequence in this order in the 5′to 3′ direction, whereas the other chain contains the F2c sequence, theF1c sequence, the 1′-th sequence, the LBc sequence and the B2 sequencein this order in the 3′ to 5′ direction. The first elongation primer(iii) contains the first elongation primer sequence, the LBc sequenceand the B2 sequence in this order in the 3′ to 5′ direction. The secondelongation primer (iv) contains the second elongation primer sequence,the F1 sequence and the F2 sequence in this order in the 3′ to 5′direction.

The FIP primer (v) contains the F2 sequence and the F1c sequence in thisorder in the 3′ to 5′ direction. The BIP primer (vi) contains the B2sequence and the complementary sequence of the 1′-th sequence in thisorder in the 3′ to 5′ direction. The loop primer (vii) contains thefifth sequence. In this example, the complementary sequence of the 1′-thsequence functions as a B1c sequence.

FIG. 15A shows an example in which the sequences of the elongationproduct (ii), the first elongation primer (iii) and the secondelongation primer (iv) are arranged in the same order as that of FIG.14A, but the third sequence is a loop primer recognition sequence.

The FIP primer (v) contains the second sequence and the 1′-th sequencein this order in the 3′ to 5′ direction. The BIP primer (vi) containsthe complementary sequence of the sixth sequence and the fifth sequencein this order in the 3′ to 5′ direction. The loop primer (vii) containsthe complementary sequence of the third sequence.

FIG. 15B shows an example in which each sequence in the example of FIG.15A set forth above is a recognition sequence to which a LAMP primer mayhybridize. In this example, the second sequence is an F2 sequence, thethird sequence is an LFc sequence, the fifth sequence is a B1c sequenceand the sixth sequence is a B2c sequence.

Therefore, as to the elongated product (ii), one chain contains the F2sequence, the LFc sequence, the complementary sequence of the 1′-thsequence, the B1c sequence and the B2c sequence in this order in the 5′to 3′ direction, whereas and the other chain contains the F2c sequence,the LF sequence, the 1′-th sequence, the B1 sequence and the B2 sequencein this order in the 3′ to 5′ direction. The first elongation primer(iii) contains the first elongation primer sequence, the B1 sequence andthe B2 sequence in this order in the 3′ to 5′ direction. The secondelongation primer (iv) contains the second elongation primer sequence,the LFc sequence and the F2 sequence in this order in the 3′ to 5′direction.

The FIP primer (v) contains the F2 sequence and the 1′-th sequence inthis order in the 3′ to 5′ direction. The BIP primer (vi) contains theB2 sequence and the B1c sequence in this order in the 3′ to 5′direction. The loop primer (vii) contains an LF sequence. In thisexample, the complementary sequence of the 1′-th sequence functions asan F1 sequence.

As described above, according to the third embodiment, a short chainnucleic acid elongation primer set which does not contain the fourthsequence and also a short chain nucleic acid detection primer set whichcontains the above-described LAMP primer set are provided. Further, anassay kit which contains a short chain nucleic acid detection primer setdescribed in the first embodiment is also provided. Moreover, the shortchain nucleic acid elongation primer set and the short chain nucleicacid detection primer set according to the second embodiment can be usedsimilarly in the short chain nucleic acid elongation method, the shortchain nucleic acid amplification method and the short chain nucleic aciddetection method described in the first embodiment.

According to the third embodiment, the second elongation primer containsthree sequences. Thus, the second elongation primer is shorter, andtherefore when used for the short chain nucleic acid elongation method,it is possible to further prevent the first elongation primer andelongated intermediate product from being formed into a secondarystructure. Moreover, the manufacturing cost of the second elongationprimer can be further reduced.

In addition, the elongated product produced using the short chainnucleic acid elongation primer set of the third embodiment contains lessprimer recognition sequences in number than the conventional elongatedproduct for the LAMP, but contains a loop primer sequence or a loopprimer recognition region as in the case of the first embodiment.Therefore, with use of the short chain nucleic acid amplification methodand the short chain nucleic acid detection method, it is possible tostably and quickly amplify the elongated product. In addition, even ifthe quantity of short chain nucleic acid is small, it can be amplifiedefficiently. Thus, with use of the short chain nucleic acid detectionprimer set of the third embodiment, it is possible to detect or quantifyshort chain nucleic acid more precisely.

The first and second elongation primers according to the thirdembodiment, if they have such a structure that the complementarysequence of the 1′-th sequence as shown in FIG. 14A or 14B is a B1csequence, a BIP primer may hybridize with the 1′-th sequence to producean amplification product when an elongated product is produced.Therefore, the nonspecific elongated product is hardly amplified. Thus,it is possible to amplify short chain nucleic acid more specifically.

The first and second elongation primers according to the thirdembodiment, if they have such a structure that the complementarysequence of the 1′-th sequence as shown in FIG. 15A or 15B is an F1sequence, an FIP primer may hybridize with the complementary sequence ofthe 1′-th sequence to produce an amplification product when an elongatedproduct is produced. Therefore, the nonspecific elongated product ishardly amplified. Thus, it is possible to amplify short chain nucleicacid more specifically.

EXAMPLES Example 1

The example in which miRNA was elongated using the short-chain nucleicacid detection primer set of the embodiment, LAMP was performed, thepresence of quantification and the specificity were evaluated will beshown hereinbelow.

Production of Short-Chain Nucleic Acid Detection Primer Set I (Example)

A short-chain nucleic acid detection primer set I having a configurationillustrated in FIG. 16 for elongating and amplifying miRNA let7-a (Table1, SEQ ID NO: 1) was produced. In the FIG. 12 , “R” represents thecorresponding DNA of miRNA let7-a (SEQ ID NO: 1), and “Rc” representsthe complementary sequence thereof. The first elongation primer containsa first elongation primer sequence, an LBc sequence, and a B2 sequence.The second elongation primer contains a second elongation primersequence, a B1c sequence, an F1 sequence, and an F2 sequence. In theelongated product obtained with this primer set, one chain contains theF2, F1, and B1c sequences, an R sequence, an LB sequence, and a B2csequence. The FIP primer contains the F2 sequence and an F1c sequence,the BIP primer contains the B2 and B1c sequences, and the loop primercontains the LB sequence.

In the design of the short-chain nucleic acid detection primer set, thesequences of the FIP primer, the BIP primer, and the loop primer (LBprimer) were determined to be SEQ ID NOS: 2, 3, and 4 illustrated inTable 1, respectively. Based on the sequences, the sequences of thefirst elongation primer (SEQ ID NO: 5) and the second elongation primer(SEQ ID NO: 6) were designed.

TABLE 1 type of  Type of oligo SEQ primer Sequence (5′ to 3′) ID NO:miRNA let7-a UGAGGUAGUAGGUUGUAUAGUU 1Short-chain nucleic acid detection primer set I FIP primerGGCCTACTGTTTATGCTCGGCTCCGGACGACTGGATCCTT 2 BIP primerGATGGGGGAAGCATCTCGGGACTTGCTGACCTGAATGGTG 3 LB primerGGCTTGCAGTTAATTGCGTAC 4 First ACTTGCTGACCTGAATGGTGTACGCAATTAACTGCAAGCC 5elongation AACTATAC primer SecondCCGGACGACTGGATCCTTAGCCGAGCATAAACAGTAGGC 6 elongationCGATGGGGGAAGCATCTCGGGGGGTGAGGTAGTAGGTTGT primer ElongatedCCGGACGACTGGATCCTTAGCCGAGCATAAACAGTAGGCC 7 productGATGGGGGAAGCATCTCGGGGGGTGAGGTAGTAGGTTGTA (positiveTAGTTGGCTTGCAGTTAATTGCGTACACCATTCAGGTCAG control) CAAGT

Elongation Reaction

A reaction solution (reaction volume: 20 μL) containing synthetic RNAlet7-a having a copy number of 10⁵, 10⁴, 10³, 10² or 0, the firstelongation primer with a final concentration of 5 nM (SEQ ID NO: 5), 67U/20 μL of reverse transcriptase (MultiScribe Reverse Transcriptase),Tris-HCl with a final concentration of 20 mM (pH 8.0), 50 mM of KCl, 8mM of MgSO₄, 10 mM of (NH₄)₂SO₄, 0.1% of Tween-20, 1.4 mM of dNTPs, 1 mMof DTT, and 4 U/20 μL of RNase OUT was used, and a reverse transcriptionreaction was performed under the following conditions: 16° C. for 30minutes, 42° C. for 30 minutes, and 85° C. for 5 minutes.

Amplification Reaction

After the completion of the reaction, to the reaction solution, thesecond elongation primer with a final concentration of 4.45 nM (SEQ IDNO: 6) and 0.4 U of DeepVent(exo-) DNA Polymerase (2 μL in total) wereadded, and the resulting mixture was subjected to elongation reaction at35 cycles of 95° C. for 20 sec, 55° C. for 30 sec, 72° C. for 30 secafter 95° C. for 2 minutes. Thereafter, the elongation reaction wasperformed at 72° C. for 5 minutes.

1 μL of the obtained reaction solution was added to a solutioncontaining the FIP primer (SEQ ID NO: 2) with a final concentration of1.6 μM, 1.6 μM of the BIP primer (SEQ ID NO: 3), 0.8 μM of the LB primer(SEQ ID NO: 4), 8 U/25 μL of Tin exo-DNA polymerase, Tris-HCl (pH 8.0),50 mM of KCl, 8 mM of MgSO₄, 10 mM of (NH₄)₂SO₄, 0.1% of Tween-20, 1.4mM of dNTPs, and 24 μL of 0.8 M betaine mixture, and the resultingmixture was incubated at 65° C. for 90 minutes for LAMP. The amplifyingwas performed using the LAMP turbidity measuring system (LT-16,manufactured by NIPPON GENE, CO., LTD.) and the time to threshold ofturbidity (hereinafter “Tt”) was measured.

The amplification reaction was performed similarly to above using anamplification reaction solution obtained by adding 10⁷ copies of anartificially synthesized product (SEQ ID NO: 7) of an elongated productto be obtained by reverse transcription and elongation of miRNA usingthe first and second elongation primers as the positive control(hereinafter, “PC”) to the above-described solution. To a negativecontrol (hereinafter, “NC”) to which no template was added, 1 μL of TEwas added and the resulting mixture was treated similarly to above. Theexperiment was performed in duplicate.

The results were shown in FIG. 17 . In the case of 10⁶ to 10² copies,there is a correlation between the abundance of miRNA and the Tt value.As the abundance of miRNA was lower, the Tt value was longer. Therefore,it is suggested that, according to the primer set of the embodiment, theelongated product is stably amplified and it is possible to quantifymiRNA.

Further, with respect to the sample not containing miRNA (“0” in thefigure), in one of the duplicate results, the turbidity rose in 67.3minutes, and in the other, no amplification occurred. The former wasassumed nonspecific amplification, and the Tt value was slower by 22minutes than the Tt value in 10² copies (41.2 minutes, 44.8 minutes,specific amplification). Therefore, it is apparent that, according tothe primer set of the embodiment, there is an obvious difference betweenthe specific amplification of 10² copies and nonspecific amplification,and it is possible to quantify miRNA even if the abundance of miRNA islow (10² copies). Consequently, it is suggested that, according to theprimer set and the method of the embodiment, it is possible toaccurately detect miRNA compared to the conventional method.

Example 2

An example will be provided below, in which using primer sets withdifferent configurations to obtain an elongated product containing sixsequences, miRNA was elongated, LAMP was performed, and the presence ofquantification and the specificity were compared.

Production of Short-Chain Nucleic Acid Detection Primer Set II (Example)

A short-chain nucleic acid detection primer set II for elongating andamplifying miRNA let7-a (Table 2, SEQ ID NO: 1) was prepared. Theshort-chain nucleic acid detection primer set II has the sameconfiguration as that illustrated in FIG. 16 , and the base sequences ofthe sequences are different from those of the short-chain nucleic aciddetection primer set I and have the base sequences illustrated in Table2. That is, the FIP primer has a base sequence of SEQ ID NO: 8, the BIPprimer has a base sequence of SEQ ID NO: 9, the loop primer (LB primer)has a base sequence of SEQ ID NO: 10, the first elongation primer has abase sequence of SEQ ID NO: 11, and the second elongation primer has abase sequence of SEQ ID NO: 12.

TABLE 2 type of  Type of oligo SEQ primer Sequence (5′ to 3′) ID NO:miRNA let7-a UGAGGUAGUAGGUUGUAUAGUU  1Short-chain nucleic acid detection primer set II FIP primerCGTCCAGCATATCAAAACCGCGCCTTCGGAGAACCCCTCT  8 BIP primerCGATTCACGATGCATCCGGCAGACGTTCTGGTACGAACTCG  9 LB primerCAACAGCAGCCGGGGAGTTG 10 First GACGTTCTGGTACGAACTCGCAACTCCCCGGCTGCTGTTGA11 elongation ACTATAC primer SecondCCTTCGGAGAACCCCTCTCGCGGTTTTGATATGCTGGACGC 12 elongationGATTCACGATGCATCCGGCAGGGTGAGGTAGTAGGTTGT primer ElongatedCCTTCGGAGAACCCCTCTCGCGGTTTTGATATGCTGGACGC 13 productGATTCACGATGCATCCGGCAGGGTGAGGTAGTAGGTTGTAT (positiveAGTTCAACAGCAGCCGGGGAGTTGCGAGTTCGTACCAGAAC control) GTC

Production of Short-Chain Nucleic Acid Detection Primer Set III(Comparative Example)

A short-chain nucleic acid detection primer set III having aconfiguration illustrated in FIG. 14 for elongating and amplifying miRNAlet7-a (Table 3, SEQ ID NO: 1) was prepared. The first elongation primercontains the first elongation primer sequence and the B2 sequence. Thesecond elongation primer contains the second elongation primer sequenceand the LB, B1c, F1, and F2 sequences. In the elongated product obtainedwith this primer set, one chain contains the F2, F1, B1c, LB, R, and B2csequences. The FIP, BIP, and loop primers are the same as those of theshort-chain nucleic acid detection primer set II.

Each of the primers of the short-chain nucleic acid detection primer setIII has each base sequence illustrated in Table 3. That is, the FIPprimer has a base sequence of SEQ ID NO: 8, the BIP primer has a basesequence of SEQ ID NO: 9, the loop primer (LB primer) has a basesequence of SEQ ID NO: 10, the first elongation primer has a basesequence of SEQ ID NO: 14, and the second elongation primer has a basesequence of SEQ ID NO: 15.

TABLE 3 type of  Type of oligo SEQ primer Sequence (5′ to 3′) ID NO:miRNA let7-a UGAGGUAGUAGGUUGUAUAGUU  1Short-chain nucleic acid detection primer set III FIP primerCGTCCAGCATATCAAAACCGCGCCTTCGGAGAACCCCTCT  8 BIP primerCGATTCACGATGCATCCGGCAGACGTTCTGGTACGAACTCG  9 LB primerCAACAGCAGCCGGGGAGTTG 10 First GACGTTCTGGTACGAACTCGAACTATAC 14 elongationprimer Second CCTTCGGAGAACCCCTCTCGCGGTTTTGATATGCTGGACGC 15 elongationGATTCACGATGCATCCGGCACAACAGCAGCCGGGGAGTTGG primer GGTGAGGTAGTAGGTTGTElongated CCTTCGGAGAACCCCTCTCGCGGTTTTGATATGCTGGACGC 16 productGATTCACGATGCATCCGGCACAACAGCAGCCGGGGAGTTGG (positiveGGTGAGGTAGTAGGTTGTATAGTTCGAGTTCGTACCAGAAC control) GTC

Elongation Reaction and Amplification Reaction

The elongation and amplification reactions were performed similarly toExample 1 using the short-chain nucleic acid detection primer sets IIand III. As positive controls, artificially synthesized elongatedproducts (SEQ ID NOS: 13 and 16, respectively) were used.

The results were shown in FIG. 19 . With respect to the short-chainnucleic acid detection primer set II, despite the fact that in one ofthe duplicate results (10² copies), no amplification occurred, there wasa correlation between the copy number of miRNA and the Tt value.Meanwhile, in the short-chain nucleic acid detection primer set III, theTt value of all the samples (including the sample not containing miRNA)was approximately 40 minutes. Therefore, it is suggested that it is notpossible to quantify miRNA using the short-chain nucleic acid detectionprimer set III. This result shows that even if the first elongationprimer and the second elongation primer are configured to obtain anelongated product containing six sequences, it is not possible toquantify miRNA when the first elongation primer contains two sequencesand the second elongation primer contains five sequences.

Example 3

The example shown hereinbelow is an example of in which using a primerset containing a dummy sequence, miRNA was elongated, LAMP wasperformed, and the presence of quantification and the specificity wereevaluated.

Production of Short-Chain Nucleic Acid Detection Primer Set IV

A short-chain nucleic acid detection primer set IV for elongating andamplifying miRNA (miR-1307-3p) (Table 4, SEQ ID NO: 17) was prepared.The short-chain nucleic acid detection primer set IV has the sameconfiguration as that illustrated in FIG. 16 , and the base sequences ofthe sequences are different from those of the short-chain nucleic aciddetection primer set I and have the base sequences illustrated in Table4. That is, the FIP primer has a base sequence of SEQ ID NO: 18, the BIPprimer has a base sequence of SEQ ID NO: 19, the loop primer (LB primer)has a base sequence of SEQ ID NO: 20, the first elongation primer has abase sequence of SEQ ID NO: 21, and the second elongation primer has abase sequence of SEQ ID NO: 22.

TABLE 4 type of Type of oligo SEQ primer Sequence (5′ to 3′) ID NO:miR-1307-3p ACUCGGCGUGGCGUCGGUCGUG 17Short-chain nucleic acid detection primer set IV FIP primerTGGAATACGTGAGACTGGGCCTAGGGTTGGAGGTTCATGTCA 18 BIP primerCTGACCACCAGTCGCACTGAGCCAGTCCGCCACTGTACT 19 LB primerAACACTGTCAGATAGGGCCTAG 20 FirstCCAGTCCGCCACTGTACTCTAGGCCCTATCTGACAGTGTTCA 21 elongation CGACCG primerSecond AGGGTTGGAGGTTCATGTCAAGGCCCAGTCTCACGTATTCCA 22 elongationCTGACCACCAGTCGCACTGAGGGGACTCGGCGTGGCGT primer ElongatedAGGGTTGGAGGTTCATGTCTlAGGCCCAGTCTCACGTATTCCA 23 productCTGACCACCAGTCGCACTGAGGGGACTCGGCGTGGCGTCGGTC (positiveGTGAACACTGTCAGATAGGGCCTAGAGTACAGTGGCGGACTGG control)

Production of Short-Chain Nucleic Acid Detection Primer Set V

A short-chain nucleic acid detection primer set V having a configurationillustrated in FIG. 19 for elongating and amplifying miRNA (miR-1307-3p)(Table 5, SEQ ID NO: 17) was produced. The first elongation primercontains the first elongation primer sequence, the dummy sequence, andthe B2 sequence. The second elongation primer contains a secondelongation primer sequence, a B1c sequence, an F1 sequence, and an F2sequence. In the elongated product obtained with this primer set, onechain contains the F2, F1, B1c, R, dummy, and B2c sequences. The FIPprimer contains the F2 and F1c sequences, the BIP primer contains the B2and B1c sequences, and the loop primer contains the R sequence.

Each of the primers of the short-chain nucleic acid detection primer setV has a base sequence illustrated in Table 5. That is, the FIP primerhas a base sequence of SEQ ID NO: 18, the BIP primer has a base sequenceof SEQ ID NO: 19, the loop primer has a base sequence of SEQ ID NO: 24,the first elongation primer has a base sequence of SEQ ID NO: 21, andthe second elongation primer has a base sequence of SEQ ID NO: 22. Theelongated product used as a positive control contains a base sequence ofSEQ ID NO: 23.

TABLE 5 type of  Type of oligo SEQ primer Sequence (5′ to 3′) ID NO:miR-1307-3p ACUCGGCGUGGCGUCGGUCGUG 17Short-chain nucleic acid detection primer set V FIP primerTGGAATACGTGAGACTGGGCCTAGGGTTGGAGGTTCATGTCA 18 BIP primerCTGACCACCAGTCGCACTGAGCCAGTCCGCCACTGTACT 19 LB primer CGTGGCGTCGGTCGTG 24First CCAGTCCGCCACTGTACTCTAGGCCCTATCTGACAGTGTTCA 21 elongation CGACCGprimer Second AGGGTTGGAGGTTCATGTCAAGGCCCAGTCTCACGTATTCCA 22 elongationCTGACCACCAGTCGCACTGAGGGGACTCGGCGTGGCGT primer ElongatedAGGGTTGGAGGTTCATGTCAAGGCCCAGTCTCACGTATTCCA 23 productCTGACCACCAGTCGCACTGAGGGGACTCGGCGTGGCGTCGGT (positiveCGTGAACACTGTCAGATAGGGCCTAGAGTACAGTGGCGGACT control) GG

Elongation Reaction and Amplification Reaction Synthetic RNA miR-1307-3phaving a copy number of 10⁶, 10⁵, 10⁴, 10³ or 0 was elongated andamplified similarly to Example 1 using the short-chain nucleic aciddetection primer sets IV and V. As a positive control, an artificiallysynthesized elongated product (SEQ ID NO: 23) was used.

The results were shown in FIG. 21 . It is suggested that, in both theshort-chain nucleic acid detection primer sets IV and V, there was acorrelation between the copy number of miRNA and the Tt value and it ispossible to quantify miRNA using those primer sets with highsensitivity. Further, in the short-chain nucleic acid detection primerset IV, the absolute value of the slope of an increase in the Tt value(in 10⁶ to 10³ copies) was 3.08, meanwhile, in the short-chain nucleicacid detection primer set V, the absolute value was 4.80. Therefore, itis apparent that it is possible to quantify miRNA accurately using theshort-chain nucleic acid detection primer set IV. Further, in theshort-chain nucleic acid detection primer set V, the Tt value innon-specific amplification with no miRNA became significantly slow(average: 38.5 minutes to 66.3 minutes). Consequently, it is suggestedthat, according to the short-chain nucleic acid detection primer set V,it is possible to achieve much higher specific detection.

Example 4

An example will be provided below, in which using primer sets which donot have a dummy sequence, miRNA was elongated, LAMP was performed, andthe presence of quantification and the specificity were compared.

Production of Short-Chain Nucleic Acid Detection Primer Sets VI(Example) and VII (Example)

Short chain nucleic acid detection primer sets VI and VII for elongatingand amplifying miRNA (miR 5p) (Table 6, SEQ ID NO: 25) were prepared.

The structure of each primer of the short chain nucleic acid detectionprimer set VI is the same as that shown in FIG. 20 , but the basesequence has that shown in Table 6. That is, the FIP primer contains SEQID NO: 26, the BIP primer contains SEQ ID NO: 27, the loop primer (LBsequence) contains SEQ ID NO: 28, the first elongation primer containsSEQ ID NO: 29, the second primer contains SEQ ID NO: 30 and theelongated product of the positive control contains a base sequence ofSEQ ID NO: 31.

The short chain nucleic acid detection primer set VII has the structureshown in FIG. 22 and contains a base sequence shown in Table 6. That is,the first elongation primer contains a first elongation primer sequence,a B2 sequence and contains a base sequence of SEQ ID NO: 32. The secondelongation primer contains a second elongation primer sequence, a B1csequence, an F1 sequence and an F2 sequence and contains a base sequenceof SEQ ID NO: 30. As to the elongated product used as a positivecontrol, one chain contains an F2 sequence, an F1 sequence, a B1csequence, an R sequence (LB sequence) and a B2c sequence and contains abase sequence of SEQ ID NO: 33.

The FIP primer, BIP primer and loop primer are the same as thosecontained in the short chain nucleic acid detection primer set VI.

TABLE 6 type of  Type of oligo SEQ primer Sequence (5′ to 3′) ID NO:miR-423-5p UGAGGGGCAGAGAGCGAGACUUU 25Short-chain nucleic acid detection primer set VI FIP primerCCGCGTGTCGTACAACTGCTACGAACACTCCGGATGGT 26 BIP primerAGGTAACGGTTCGACCTCGAGGGGAGGTCCCTCTTTTA 27 AGCG LB primerGGGGCAGAGAGCGAGACTTT 28 First GGAGGTCCCTCTTTTAAGCGCTGTTGGATCCGCCGCTA 29elongation AGTCTCG primer Second ACGAACACTCCGGATGGTAGCAGTTGTACGACACGCGG30 elongation AGGTAACGGTTCGACCTCGAGGGGGTGAGGGGCAGAGA primer G ElongatedACGAACACTCCGGATGGTAGCAGTTGTACGACACGCGG 31 productAGGTAACGGTTCGACCTCGAGGGGGTGAGGGGCAGAGA (positiveGCGAGACTTTGCGGCGGATCCAACAGCGCTTAAAAGAG control) GGACCTCCShort-chain nucleic acid detection primer set VII FIP primerCCGCGTGTCGTACAACTGCTACGAACACTCCGGATGGT 26 BIP primerAGGTAACGGTTCGACCTCGAGGGGAGGTCCCTCTTTTA 27 AGCG LB primerGGGGCAGAGAGCGAGACTTT 28 First GGAGGTCCCTCTTTTAAGCGAAAGTCTCG 32elongation primer Second ACGAACACTCCGGATGGTAGCAGTTGTACGACACGCGGA 30elongation GGTAACGGTTCGACCTCGAGGGGGTGAGGGGCAGAGAG primer ElongatedACGAACACTCCGGATGGTAGCAGTTGTACGACACGCGGA 33 productGGTAACGGTTCGACCTCGAGGGGGTGAGGGGCAGAGAGC (positiveGAGACTTTCGCTTAAAAGAGGGACCTCC control)

Elongation Reaction

Using the short chain nucleic acid detection primer sets VI and VII, acopy number of 10⁶, 10⁵, 10⁴, 10³ or 0 of synthetic RNA miR-423-5p wereelongated and amplified. Then, using a reaction mixture (reactionvolume: 20 μL) containing the first elongation primer (SEQ ID NOS: 29and 32) (final concentration: 10 nM), 67 U/20 μL of reversetranscriptase (MultiScribe Reverse Transcriptase), RT buffer (lx) inHigh-Capacity cDNA Reverse Transcription Kit, dNTPs (finalconcentration: 0.1 mM) and 4 U/20 μL of RNase OUT, the reversetranscription reactions were carried out under the following conditions:16° C. for 10 minutes, 42° C. for 5 minutes and 85° C. for 5 minutes.

Amplification Reaction

After the completion of each reaction, the second amplification primer(SEQ ID NO: 30) (final concentration: 10 nM) and 0.4 U of DeepVent(exo-)DNA Polymerase (a total of 5 μL) were added to the reaction mixture, andthe mixture was subjected to the elongation reaction at 20 cycles of 95°C. for 20 sec, 55° C. for 30 sec, and 72° C. for 30 sec after 95° C. for2 minutes. Thereafter, the elongation reaction was carried out at 72° C.for 5 minutes.

1 μL of each of the obtained reaction mixtures was added to a solutioncontaining the FIP primer (SEQ ID NO: 26) (final concentration: 1.6 μM),1.6 μM of the BIP primer (SEQ ID NO: 27), 0.8 μM of the LB primer (SEQID NO: 28), 8 U/25 μL of Tin exo-DNA polymerase, Tris-HCl (pH 8.0), 50mM of KCl, 8 mM of MgSO₄, 10 mM of (NH₄)₂SO₄ and 0.1% of Tween-20, 1.4mM of dNTPs and 24 μL of betaine mixed-solution (0.8 M), and the mixturewas subjected to the LAMP under the conditions of 65° C. for 90 minutes.The amplification was carried out using the endpoint turbidity measuringsystem (LT-16, manufactured by NIPPON GENE, CO., LTD.) and the Tt valuewas measured. As positive controls, artificially synthesized elongatedproducts (SEQ ID NOS: 31 and 33) were used.

The results are shown in FIG. 23 . In both short chain nucleic aciddetection primer sets VI and VII, a correlation was found between thecopy number of miRNA and the Tt value. In the short chain nucleic aciddetection primer set VII, non-specific amplification which does notcontain miRNA was observed in one of two series, which was acceptablesince it was sufficiently later than that of the specific amplification.Thus, it was suggested that even with use of the primer set VII whichdoes not contain a dummy sequence, miRNA can be quantified with highsensitivity.

While certain embodiments have been described, these embodiments havebeen presented by way of example only, and are not intended to limit thescope of the inventions. Indeed, the novel embodiments described hereinmay be embodied in a variety of other forms; furthermore, variousomissions, substitutions and changes in the form of the embodimentsdescribed herein may be made without departing from the spirit of theinventions. The accompanying claims and their equivalents are intendedto cover such forms or modifications as would fall within the scope andspirit of the inventions.

What is claimed is:
 1. A primer set comprising: a short-chain nucleicacid elongation primer set and an amplification primer set, wherein theshort-chain nucleic acid elongation primer set is configured to yield anelongated product from a target short-chain RNA containing a firstsequence, the elongated product is a mutually complementarydouble-stranded nucleic acid, one chain contains a second sequence, athird sequence, a fourth sequence, a complementary sequence of cDNA ofthe first sequence (hereinafter, “cDNA of the first sequence” referredto as “1′-th sequence”), fifth sequence and a sixth sequence in thisorder in a 3′ to 5′ direction, the short-chain nucleic acid elongationprimer set comprises: a first elongation primer comprising a firstelongation primer sequence which hybridizes with the first sequence, acomplementary sequence of the fifth sequence and a complementarysequence of the sixth sequence in this order in the 3′ to 5′ direction;and a second elongation primer comprising a second elongation primersequence which hybridizes with the 1′-th sequence, the fourth sequence,the third sequence, and the second sequence in this order in the 3′ to5′ direction, and the amplification primer set is configured to amplifythe elongated product by LAMP method which comprises; the FIP primercomprising the second sequence and a complementary sequence of the thirdsequence in this order in the 3′ to 5′ direction, the BIP primercomprising the complementary sequence of the sixth sequence and thefourth sequence in this order in the 3′ to 5′ direction, and the loopprimer comprising the complementary sequence of the 1′-th sequence. 2.The primer set of claim 1, wherein: the second sequence is an F2sequence, the third sequence is an F1 sequence, the fourth sequence is aBic sequence, the fifth sequence is a dummy sequence, the sixth sequenceis a B2c sequence, the first elongation primer comprises the firstelongation primer sequence, a complementary sequence of the dummysequence, and a B2 sequence in this order in the 3′ to 5′ direction, thesecond elongation primer comprises the second elongation primersequence, the B1c sequence, the F1 sequence, and the F2 sequence in thisorder in the 3′ to 5′ direction, the FIP primer containing the F2sequence and an F1c sequence in this order in the 3′ to 5′ direction,the BIP primer containing the B2 sequence and the B1c sequence in thisorder in the 3′ to 5′ direction, the loop primer containing thecomplementary sequence of the 1′-th sequence, the F1 sequence iscomplementary to the F1c sequence, the B2 sequence is complementary tothe B2c sequence, and the dummy sequence is a base sequence differentfrom the first to sixth sequences and the complementary sequencesthereof.
 3. An assay kit, comprising: the primer set of claim 1, anucleic acid elongation reagent, and a LAMP reagent.
 4. The assay kit ofclaim 3, wherein the short-chain nucleic acid elongation primer set andthe amplification primer set in the short-chain nucleic acid detectionprimer set are accommodated in separate containers.
 5. The assay kit ofclaim 3, further comprising: a marker substance generating an electricalsignal which changes with an increase in the amplification product; andan electrochemical detection device for detecting the electrical signal.6. A short-chain nucleic acid detection method for detecting a targetshort-chain nucleic acid containing a first sequence in a sample usingthe primer set of claim 1, the method comprising: hybridizing the firstelongation primer with the first sequence and elongating the firstsequence to obtain an elongated intermediate product containing the1′-th sequence; dissociating the elongated intermediate product from thetarget short-chain nucleic acid; hybridizing the second elongationprimer with the 1′-th sequence of the elongated intermediate product andelongating the second elongation primer and the elongated intermediateproduct to obtain the elongated product; maintaining an amplificationreaction solution containing the elongated product, the amplificationprimer set, and a strand displacement DNA polymerase under isothermalamplification reaction conditions, thereby amplifying the F-th sequenceand/or the complementary sequence thereof using the elongated product asa template to obtain an amplification product; and detecting theamplification product during maintaining under the isothermalamplification reaction conditions.
 7. The method of claim 6, wherein theamplification reaction solution comprises the marker substancegenerating an electrochemical signal which changes with an increase inthe amplification product, and the detecting is performed by detectingthe electrochemical signal.
 8. The method of claim 6, wherein the secondsequence is an F2 sequence, the third sequence is an F1 sequence, thefourth sequence is a B1c sequence, the fifth sequence is a dummysequence, the sixth sequence is a B2c sequence, the first elongationprimer comprises the first elongation primer sequence, a complementarysequence of the dummy sequence, and a B2 sequence in this order in the3′ to 5′ direction, the second elongation primer comprises the secondelongation primer sequence, the B1c sequence, the F1 sequence, and theF2 sequence in this order in the 3′ to 5′ direction, the FIP primercontaining the F2 sequence and an F1c sequence in this order in the 3′to 5′ direction, the BIP primer containing the B2 sequence and the B1csequence in this order in the 3′ to 5′ direction, the loop primercontaining the complementary sequence of the 1′-th sequence, the F1sequence is complementary to the F1c sequence, the B2 sequence iscomplementary to the B2c sequence, and the dummy sequence is a basesequence different from the first to sixth sequences and thecomplementary sequences thereof, and the method further comprisingamplifying the elongated product to obtain a amplification product witha LAMP primer set comprising the FIP primer containing an F2 sequenceand an F1c sequence in this order in the 3′ to 5′ direction, the BIPprimer comprising a B2 sequence and a B1c sequence in this order in the3′ to 5′ direction, and the loop primer comprising g the complementarysequence of the 1′-th sequence, and the F1c sequence is complementary tothe F1 sequence and the B1c sequence is complementary to the B1sequence.